Selective enrichment media and uses thereof

ABSTRACT

Selective enrichment media and methods for selectively growing and detecting  Salmonella  spp. and/or Shiga toxin-producing  E. coli . The media may comprise a carbon and nitrogen source, an inorganic salt, a fermentable sugar, one or more selective agents, and an efflux pump inhibitor. Various selective agents include sulfa drugs, surfactants, aminocoumarins, cycloheximide, supravital stains, ascorbic acid, bromobenzoic acid, myricetin, nitrofurantoin, rifamycins, polyketides, and oxazolidinones. Various efflux pump inhibitors include arylpiperazines, such as 1-(1-naphthylmethyl)piperazine, and quinoline derivatives, such as 4-chloroquinoline. Methods of selectively growing and detecting  Salmonella  and/or Shiga toxin-producing  E. coli  are provided.

FIELD OF THE INVENTION

The invention is directed to selective enrichment and indicator media.The invention is more specifically directed to the use of bacterialefflux pump inhibitors to enhance the selectivity of selectiveenrichment media for the isolation and detection of certainmicroorganisms, such as Salmonella species and/or Shiga toxin-producingE. coli strains.

BACKGROUND

Foodborne salmonellosis is a major public health problem. Salmonella isthe second leading cause of annual foodborne illness cases, with anestimated 1,340,000 cases compared with nearly 2,000,000 forCampylobacter. A survey conducted in the United States estimated that in1999 non-typhoidal salmonellosis of foodborne origin causedapproximately 15,600 hospitalizations and 550 deaths (Mead, P. S., L.Slutsker, V. Dietz, et al. Food-related illness and death in the UnitedStates. Emerg. Infect. Dis. 5:607-625. 1999). Although the genusSalmonella has more than 2300 serovars, only a relatively restrictednumber belonging mainly to the Enterica subspecies of the Salmonellaenterica species are responsible for the great majority of humaninfections. The most common sources of Salmonella infections in humansare contaminated foods, including eggs, poultry, produce, meat, and meatproducts. Eggs and poultry meat are recognized as the major vehicles ofhuman infections because of epizootics in fowl.

Early detection of foodborne Salmonella is vital for food safetyassurance. However, conventional methods for detecting foodborneSalmonella are laborious and time consuming. These methods typicallyinvolve identifying presumptively positive samples by sequentiallyprocessing samples in a pre-enrichment phase, a selective-enrichmentphase, and then an analysis phase. The analysis phase may involveculturing the enriched sample on selective differential agar, analyzingwith polymerase chain reaction (PCR), and/or analyzing via immunoassay.After identifying the presumptively positive samples, confirmation ofthe presumptively positive samples typically requires biochemicalcharacterization of isolates obtained from selective, differential agarmedia. See, e.g., U.S. Food and Drug Administration (2011)Bacteriological Analytical Manual, Chapter 5. It is estimated thatmillions of such Salmonella analyses are run routinely in the UnitedStates each year.

Early detection of Salmonella is needed if foodborne illnesses caused bySalmonella are to be reduced. While determinative microbiology requiresconfirmation of presumptively positive samples, this is not the case inmany applications such as environmental monitoring and food safety-HACCPtesting. In such applications, only a reasonable presumption that asample is contaminated is required to take such corrective actions asmodifying a sanitation procedure or quarantining a product lot pendingsubsequent confirmation. Rapid screening tests which have comparablediagnostic performance to culture methods or other rapid molecularmethods can suffice.

Several Salmonella-selective enrichment media and systems are known inthe art. See, e.g., U.S. Pat. No. 4,279,995 to Woods et al.; U.S. Pat.No. 5,208,150 to Tate et al.; U.S. Food and Drug Administration (2011)Bacteriological Analytical Manual, Chapter 5; U.S. Pat. No. 7,704,706 toDruggan; U.S. Pat. No. 7,150,977 to Restaino; U.S. Pat. No. 6,368,817 toPerry et al.; U.S. Pat. No. 5,434,056 to Monget et al.; and U.S. Pat.No. 5,194,374 to Rambach. The media and systems described in thesereferences do not provide sufficient selectivity of Salmonella spp.

Another major cause of foodborne illness is Shiga toxin-producing E.coli (STEC). STEC has been linked with the severe complicationhemolytic-uremic syndrome (HUS) and other deleterious outcomes. Shigatoxin-producing E. coli is known by a number of other names, includingenterohemorrhagic E. coli (EHEC), Shiga-like toxin-producing E. coli(SLTEC), hemolytic uremic syndrome-associated enterohemorrhagic E. coli(HUSEC), and verocytotoxin- or verotoxin-producing E. coli (VTEC).

All the Shiga toxin-producing E. coli strains produce Shiga toxin (alsoknown as, Shiga-like toxin or verotoxin), a major cause of foodborneillness. EHEC strains are distinguished from commensal E. coli. Shigatoxin-producing E. coli strains can be distinguished from non-Shigatoxin-producing E. coli strains by detecting the Shiga toxin genes (Stx1and/or Stx2) in E. coli or the gene products thereof. See, e.g.,Chassagne L, Pradel N, Robin F, Livrelli V, Bonnet R, Delmas J.Detection of stx1, stx2, and eae genes of enterohemorrhagic Escherichiacoli using SYBR Green in a real-time polymerase chain reaction. DiagnMicrobiol Infect Dis. 2009 May; 64(1):98-101.

The best known of Shiga toxin-producing E. coli strains is O157:H7, butnon-O157 strains cause an estimated 36,000 illnesses, 1,000hospitalizations and 30 deaths in the United States yearly. Food safetyspecialists recognize the “Big Six” Shiga toxin-producing E. colistrains: O26, O45, O103, O111, O121, and O145. A 2011 outbreak inGermany was caused by another Shiga toxin-producing E. coli strain,O104:H4. This strain has both enteroaggregative and enterohemorrhagicproperties. Both the O145 and O104 strains can cause hemolytic-uremicsyndrome. The former strain was shown to account for 2% to 51% of knownHUS cases. An estimated 56% of such cases are caused by O145 and 14% byother Shiga toxin-producing E. coli strains.

Shiga toxin-producing E. coli strains that induce bloody diarrhea leadto HUS in 10% of cases. The clinical manifestations of postdiarrheal HUSinclude acute renal failure, microangiopathic hemolytic anemia, andthrombocytopenia. The Shiga toxin can directly damage renal andendothelial cells. Thrombocytopenia occurs as platelets are consumed byclotting. Hemolytic anemia results from intravascular fibrin deposition,increased fragility of red blood cells, and fragmentation.

An important trend in the meat industry in the United States is theability to cultivate E. coli and Salmonella in the same selectiveenrichment media. Laboratories would like to streamline their processesby reducing the steps to regulatory compliance to one operation forscreening for both pathogenic E. coli and Salmonella.

There exists a need for a simple, rapid screening test that identifiespresumptively positive Salmonella and/or Shiga toxin-producing E. colisamples at an early stage of the sample analysis, preferably in aselective enrichment stage of the analysis. Simultaneous enrichment anddetection of Salmonella and/or Shiga toxin-producing E. coli using asingle testing method would reduce not only time but also the cost oflabor and media. Streamlining procedures and reducing labor and testcosts should permit more frequent monitoring for Shiga toxin-producingE. coli, thereby reducing contamination hazard.

SUMMARY OF THE INVENTION

The present invention provides selective enrichment media for Salmonellaspecies such as Salmonella enterica, and/or Shiga toxin-producing E.coli. The present invention also provides selective indicator brothsthat simultaneously enrich and detect Salmonella and/or Shigatoxin-producing E. coli within a sample. The selective indicator brothsincorporate metabolic indicators that produce a visually detectablesignal, such as a color change, as Salmonella species or Shigatoxin-producing E. coli strains grow in the media and ferment thesubstrates or optionally hydrolyze chromogenic substrates such as Xgal,etc. The media of the invention incorporate selectively targetedinhibitors to prevent growth by background micro-flora and, hence,metabolism of the indicator substrates, thereby preventing undesirablefalse positive responses.

The present invention provides selective enrichment media capable ofsuppressing the growth of Klebsiella, Enterobacter, Citrobacter, andcommensal E. coli species without compromising the growth of Salmonellaenterica and/or Shiga toxin-producing E. coli by incorporating effluxpump inhibitors (EPIs) in the media. One of the principal mechanisms bywhich bacteria have evolved to defeat toxic compounds in the environmentis the presence of proteins in bacterial membranes capable of pumpingthese compounds out of the intracellular space. These proteins, known asefflux pumps, reside in both cytoplasmic and outer membrane structuresof certain bacteria (Piddock, L. J. Clinically Relevant ChromosomallyEncoded Multidrug Resistance Efflux Pumps in Bacteria. Clin. Microbiol.Rev. 19(2): 382-402. 2006). Various classes of both natural andsynthetic compounds can inhibit these efflux pumps. Some inhibitors arehighly active across the Enterobacteriacae family in preventing theejection of numerous antibiotics. For example, the peptidomimeticcompound Phe-Arg-β-naphthylamide has been demonstrated to be effectivein many gram-negative bacteria, including E. coli and Salmonellaenterica (Piddock, L. J. Clinically Relevant Chromosomally EncodedMultidrug Resistance Efflux Pumps in Bacteria. Clin. Microbiol. Rev.19(2): 382-402. 2006). Some of the arylpiperazines and alkylquinolinesappear to be more limited in their spectrum of action (Bohnert, J. etal. Selected Arylpiperazines Are Capable of Reversing MultidrugResistance in Escherichia coli Overexpressing RND Efflux Pumps.Antimicrob. Agents & Chemoth. 49(2):849-852. 2005; Schumacher, A. et al.Effect of 1-(1-naphthylmethyl)-piperazine, a novel putative efflux pumpinhibitor, on antimicrobial drug susceptibility in clinical isolates ofEnterobacteriacae other than E. coli. J. Antimicrob. Chemoth. 57:344-348. 2005; and Mahamoud, A. et al. Antibiotic efflux pumps inGram-negative bacteria: the inhibitor response strategy. J. Antimicrob.Chemoth. 59(6):1223-1229. 2007).

The present invention employs antibiotics and EPIs that enable selectiveenrichment for Salmonella and/or Shiga toxin-producing E. coli. It hasbeen found that neither arylpiperazines nor alkylquinolines increase thesusceptibility of Salmonella enterica or Shiga toxin-producing E. colito the several antibiotics known to be rendered effective against othergram negative bacteria such as Enterobacter spp. and commensal E. coli.The resistance of Salmonella enterica and Shiga toxin-producing E. colito the antibiotic-sensitizing effects of arylpiperazines andalkylquinolines forms the basis of at least some versions of theselective enrichment media of the present invention. These media aremore selective for Salmonella than those currently in use, such asRapaport-Vasiliadis medium and tetrathionate broth (U.S. Food and DrugAdministration (2011) Bacteriological Analytical Manual, Chapter 5).

An exemplary selective enrichment medium of the invention comprises acarbon and nitrogen source, an inorganic salt, a selective agent, and anefflux pump inhibitor. The selective agent preferably comprises one ormore of a sulfa drug, a surfactant, an aminocoumarin, cycloheximide,myricetin, nitrofurantoin, a rifamycin, a polyketide, and anoxazolidinone.

The efflux pump inhibitor may comprise one or more of a phenothiazine, aphenylpiperidine, a tetracycline analog, an aminoglycoside analog, afluoroquinolone analog, a quinoline derivative, a peptidomimetic, apyridopyrimidine, an arylpiperidine, and an arylpiperazine. The effluxpump inhibitor preferably comprises one or more of an arylpiperazine anda quinoline derivative. In some versions, the efflux pump inhibitor maycomprise 1-(1-naphthylmethyl)piperazine. In some versions, the effluxpump inhibitor may comprise 4-chloroquinoline.

Some media include 2-deoxy-D-Ribose as a fermentable sugar.

Some media include one or more of sulfanilamide and sulfathiazole as asulfa drug. Sulfanilamide and sulfathiazole may be included in the mediain a ratio of about 9:1.

Some media include 7-ethyl-2-methyl-4-undecyl sulfate or a salt thereofas a selective agent.

Some media include novobiocin as an aminocoumarin.

Some media include rifampicin as a rifamycin.

Some media include one or more of tetracycline and doxycycline as apolyketide.

Some media include linezolid as an oxazolidinone.

Some media include a fermentable sugar. The fermentable sugar mayinclude 2-deoxy-D-Ribose.

Some media include a supravital stain. The supravital stain may comprisebrilliant green.

Some media include ascorbic acid.

Some media include bromobenzoic acid.

Some media include one or more of a rifamycin, a polyketide, and anoxazolidinone as a selective agent, and one or more of an arylpiperazineand a quinoline derivative as an efflux pump inhibitor.

Some media include one or more of rifampicin, tetracycline, doxycycline,and linezolid as a selective agent, and one or more of1-(1-naphthylmethyl)piperazine and 4-chloroquinoline as an efflux pumpinhibitor.

Some media include a sulfa drug, a surfactant, an aminocoumarin,cycloheximide, myricetin, and nitrofurantoin as a selective agent, andfurther include one or more of an arylpiperazine and a quinolinederivative as an efflux pump inhibitor.

Some media include a sulfa drug, a surfactant, an aminocoumarin,cycloheximide, myricetin, nitrofurantoin, and one or more of arifamycin, a polyketide, and an oxazolidinone as a selective agent, andfurther include one or more of an arylpiperazine and a quinolinederivative as an efflux pump inhibitor.

Some media include a visual indicator that indicates the presence of amicroorganism selected from the group consisting of Salmonella and E.coli.

The selective agent and the efflux pump inhibitor may be present inamounts effective to inhibit growth of at least one non-Salmonellaspecies to a greater extent than one or more Salmonella species. Theselective agent and the efflux pump inhibitor are also preferablypresent in amounts effective to inhibit growth of at least onenon-Salmonella species while not substantially inhibiting growth of oneor more Salmonella species.

The selective agent and the efflux pump inhibitor may be present inamounts effective to inhibit growth of at least one non-Shigatoxin-producing E. coli strain to a greater extent than one or moreShiga toxin-producing E. coli strains. The selective agent and theefflux pump inhibitor are also preferably present in amounts effectiveto inhibit growth of at least one non-Shiga toxin-producing E. colistrain while not substantially inhibiting growth of one or more Shigatoxin-producing E. coli strains.

The components of the media are preferably present in amounts thatpermit growth of E. coli having a type selected from O157, O145, O104,O26, O111, O103, and O91.

The media of the invention may be in a liquid form, i.e., a broth, ormay be in solid or semi-solid form, e.g., agar-based.

The invention also provides for methods of selectively enriching for amicroorganism selected from the group consisting of Salmonella and Shigatoxin-producing E. coli. The methods comprise culturing a samplesuspected of containing Salmonella and/or Shiga toxin-producing E. coliin selective enrichment media as described herein. The culturingpreferably grows Salmonella, Shiga toxin-producing E. coli, or bothSalmonella and Shiga toxin-producing E. coli, such as at least oneSalmonella species, at least one Shiga toxin-producing E. coli strain,or at least one Salmonella species and at least one Shigatoxin-producing E. coli strain. In some versions, the culturing growsSalmonella enterica. In some versions, the culturing grows E. colihaving a type selected from O157, O145, O104, O26, O111, O103, and O91.Further, the culturing preferably substantially inhibits growth of amicroorganism selected from the group consisting of Citrobacter spp. andnon-Shiga toxin-producing E. coli, such as at least one Citrobacter sp.and/or at least one non-Shiga toxin-producing E. coli strain. Themethods may further comprise detecting presence of the microorganism,such as Salmonella, Shiga toxin-producing E. coli, or both Salmonellaand Shiga toxin-producing E. coli, such as at least one Salmonellaspecies, at least one Shiga toxin-producing E. coli strain, or at leastone Salmonella species and at least one Shiga toxin-producing E. colistrain.

The objects and advantages of the invention will appear more fully fromthe following detailed description of the preferred embodiment of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

The various components of the media of the present invention aredescribed as follows.

The media of the invention preferably include a carbon and nitrogensource. Preferred carbon and nitrogen sources include proteinhydrolysates and/or extracts. Suitable carbon and nitrogen sourcesinclude peptone, neopeptone, tryptone beef extract paste, desiccatedpowder of beef heart, desiccated powder of beef liver, brain heartinfusion, digests of casein, and yeast extract. Examples include thefollowing products from BD (Franklin Lakes N.J.): ACIDICASE™ Peptone(hydrochloric acid hydrolysis of casein; Cat. No. 211843); Beef ExtractPaste (Cat. No. 212610); Beef Heart for Infusion (Desiccated powder ofbeef heart, Cat. No. 213210); BIOSATE™ Peptone (Cat No. 211862);BIOSATE™ Peptone (Cat. No. 294312); Brain Heart Infusion (Cat. No.237300); Casamino acids (acid hydrolyzed casein; Cat. Nos. 223050,223020, 223120, 223030, 223110, 228820, 228830, and 228830); CaseinDigest (Enzymatic digest of casein for molecular genetics, Cat. No.211610); Casitone (Pancreatic digest of casein; Cat. Nos. 225930 and225910); Gelatin (Cat. Nos. 214340 and 214320); GELYSATE™ Peptone(Pancreatic digest of gelatin; Cat. No. 211870); Liver (Desiccatedpowder of beef liver; Cat. No. 213320); Neopeptone (Enzymatic digest ofprotein; Cat. Nos. 211680 and 211681); Peptone (An enzymatic digest ofprotein; Cat Nos. 211830, 211677, 254820, 211820); PHYTONE™ Peptone (Anenzymatic digest of soybean meal, Non-animal origin; Cat. Nos. 211906and 298147); PHYTONE™ Peptone UF (Ultra-filtered enzymatic digest ofsoybean meal, designed specifically for cell culture applications,non-animal origin; Cat. Nos. 210931 and 210936); Polypeptone Peptone(Pancreatic digest of casein and peptic digest of animal tissue combinedin equal parts; Cat. Nos. 211910 and 297108); Proteose Peptone(Enzymatic digest of protein, high in proteoses; Cat. Nos. 212010,253310, and 211684); Proteose Peptone No. 2 (Enzymatic digest ofprotein; Cat. Nos. 212120 and 212110); Proteose Peptone No. 3 (Enzymaticdigest of protein; Cat. Nos. 211693, 211692, 212220, and 212230)Proteose Peptone No. 4 (Enzymatic digest of protein; Cat. No. 211715);Select Soytone (Enzymatic digest of soybean meal, non-animal origin;Cat. Nos. 212489 and 212488); Soytone, BACTO™ (Enzymatic hydrolysate ofsoybean meal; Cat. Nos. 243620 and 243610); TC Yeastolate (Water solubleportion of autolyzed yeast, source of Vitamin B complex, tested fortissue culture; Cat. Nos. 255772 and 255771); TRYPTICASE™ Peptone(Enzymatic digest of casein; Cat. No. 211921); TRYPTICASE™ Peptone(Enzymatic digest of casein; Cat. Nos. 211922 and 211923); Tryptone(Enzymatic digest of casein; Cat. Nos. 211705, 211701, and 211699);Tryptone, BITEK™ (Enzymatic digest of casein; Cat. No. 251420); Tryptose(Enzymatic hydrolysate of protein; Cat. Nos. 211709 and 211713); YeastExtract (Water-soluble extract of autolyzed yeast cells suitable for usein culture media; Cat. Nos. 212720, 211931, 211929, 212710, 212730,211930, and 212750); Yeast Extract, LD (Water-soluble extract ofautolyzed yeast cells that has been agglomerated to minimize dusting;Cat. Nos. 210933 and 210941); Yeast Extract, UF (Water-soluble extractof autolyzed yeast cells, ultra-filtration enhances solubility andlowers the endotoxin, suitable for use in cell culture and microbialfermentation; Cat. Nos. 210934 and 210929); and equivalents thereof.Peptone or tryptone supplemented with beef or yeast extract arepreferred carbon and nitrogen sources. Suitable concentration ranges ofthe carbon and nitrogen source are from about 1 g/L to about 300 g/L,preferably about 2 g/L to about 150, and most preferably from about 10g/L to about 30 g/L.

The media of the invention may include an inorganic salt. Suitableinorganic salts include calcium salts, copper salts, iron salts,selenium salts, potassium salts, magnesium salts, sodium salts, ammoniumsalts, nickel salts, tin salts, and zinc salts, among others. Suitableexamples of such salts include CaCl₂, CuSO₄, FeSO₄, H₂SeO₃, KCl, KI,KH₂PO₄, MgCl₂, MgCO₃, MgSO₄, MnSO₄, Na₂HPO₄, Na₂SiO₃, NaCl, NaH₂PO₄,NaHCO₃, NH₄VO₃, (NH₄)₆Mo₇O₂₄, NiCl₂, SnCl₂, ZnSO₄, and hydrates thereof.Magnesium, potassium, calcium, iron and/or zinc salts are preferred.Magnesium salts, such as magnesium chloride (MgCl₂), magnesium carbonate(MgCO₃), magnesium sulfate (MgSO₄), and hydrates thereof areparticularly preferred. A particularly suitable magnesium salt ismagnesium chloride. The inorganic salt is preferably included at aconcentration sufficient to create high osmotic pressure in the medium.Suitable concentration ranges of the inorganic salt are from about 0.1g/L to about 50 g/L, preferably about 2 g/L to about 40 g/L, morepreferably from about 4 g/L to about 35 g/L, and most preferably fromabout 6 g/L to about 15 g/L.

The media of the invention may include a pH indicator. The pH indicatoris preferably sensitive to acidification. The pH indicator is preferablyan indicator that transitions color in a range of about pH 7 to about pH5. Examples of suitable pH indicators are bromocresol purple, phenolred, and neutral red. Bromocresol purple is preferred. The pH indicatormay be included in any concentration suitable for detecting the pHchange. Exemplary ranges include about 0.004 g/L to about 0.25 g/L, suchas about 0.02 g/L to about 0.05 g/L.

The media of the invention may include a fermentable sugar. Examples ofsuitable sugars include adonitol, arabinose, arabitol, ascorbic acid,5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), chitin,D-cellubiose, 2-deoxy-D-ribose, dulcitol, (S)-(+)-erythrulose, fructose,fucose, galactose, glucose, isopropyl β-D-1-thiogalactopyranoside(IPTG), inositol, lactose, lactulose, lyxose, maltitol, maltose,maltotriose, mannitol, mannose, melezitose, melibiose, microcrystallinecellulose, palatinose, pentaerythritol, raffinose, rhamnose, ribose,sorbitol, sorbose, starch, sucrose, trehalose, xylitol, xylose, andhydrates thereof. For selection of Salmonella spp., it is preferred touse a sugar that can be efficiently metabolized by Salmonella spp. butnot by other bacteria. 2-Deoxy-D-ribose, xylose, mannitol, dulcitol,sorbitol, L-rhamnose and D-arabitol are suitable for this purpose.2-Deoxy-D-ribose is particularly preferred because of its relativeselectivity toward Salmonella enterica. It was previously thought that,with the exception of a few Citrobacter species, most non-Salmonellaspecies within Enterobacteriacae are incapable of fermenting2-deoxy-D-ribose (Tourneux, L. et al. Genetic and BiochemicalCharacterization of Salmonella enterica Serovar Typhi Deoxyribokinase.J. Bact. 182(4):869-873. 2000; and Christensen, M. et al. Regulation ofExpression of the 2-Deoxy-D-Ribose Utilization Regulon, deoQKPX, fromSalmonella enterica Serovar Typhimurium. J. Bact. 185(20):6042-6050.2003). However, more recent publications have provided evidence thatsome Klebsiella and Enterobacter species can also ferment2-deoxy-D-ribose (Hansen et al. Recommended Test Panel forDifferentiation of Klebsiella Species on the Basis of a TrilateralInterlaboratory Evaluation of 18 Biochemical Tests. J. Clin. Microbiol.42(8):3665-3669. 2004). The fermentable sugar may be included in themedia at a concentration of from about 0.5 g/L to about 120 g/L,preferably of from about 1.0 g/L to about 60 g/L, and more preferably offrom about 5.0 to about 12.0 g/L.

The media of the invention may include one or more visual indicators.“Visual indicators” as used herein denote components that provide avisual indication of the presence of one or more types ofmicroorganisms. The media of the invention preferably include one ormore visual indicators that indicate the presence of Salmonella; E.coli, such as Shiga toxin-producing E. coli; or both Salmonella and E.coli. Exemplary indicators include the combination of bromocresol purpleand 2-deoxy-D-ribose for indicating the presence of Salmonella, and thecombination of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal)and isopropyl β-D-1-thiogalactopyranoside (IPTG) for indicating thepresence of E. coli and other lactose-positive coliforms. Other visualindicators for indicating the presence of Salmonella, E. coli, and othermicroorganisms are known in the art.

The media of the invention may include one or more selective agentsdescribed below, or otherwise known in the art. Each selective agent maybe included in an amount effective to inhibit growth of at least onenon-Salmonella microorganism and/or at least one non-Shigatoxin-producing E. coli microorganism to a greater extent thanSalmonella (such as Salmonella enterica) and/or Shiga toxin-producing E.coli (such as E. coli having a type selected from O157, O145, O104, O26,O111, O103, and O91). It is preferred that the one or more selectiveagents are present in amounts that do not substantially inhibit thegrowth or metabolism of Salmonella (such as Salmonella enterica) and/orShiga toxin-producing E. coli (such as E. coli having a type selectedfrom O157, O145, O104, O26, O111, O103, and O91).

The media of the invention may include one or more sulfa drugs as aselective agent. The sulfa drug serves as an anti-metabolite selectiveagent. Sulfa drugs, also called sulfonamides or sulphonamides, areantimicrobial agents that contain the sulfonamide group. Examples ofsuitable sulfa drugs include aldesulfone sodium, elixir sulfanilamide,mafenide, phthalylsulfathiazole, prontosil, silver sulfadiazine,succinylsulfathiazole, sulfabenzamide, sulfacetamide, sulfacytine,sulfadiazine, sulfadicramide, sulfadimethoxine, sulfadimidine,sulfadoxine, sulfafurazole, sulfaguanidine, sulfalene, sulfamazone,sulfamerazine, sulfamethizole, sulfamethoxazole, sulfamethoxypyridazine,sulfametomidine, sulfametoxydiazine, sulfametrole, sulfamoxole,sulfanilamide, sulfaperin, sulfaphenazole, sulfapyridine,sulfaquinoxaline, sulfathiazole, sulfathiourea, sulfatolamide, andsulfisomidine, among others. Sulfathiazole is preferably includedbecause of its toxicity to some Citrobacter spp. A preferred combinationof sulfa drugs includes sulfanilamide and sulfathiazole in a ratio ofabout 9:1, such as in concentrations of about 0.9 g/L and 0.1 g/L,respectively. The one or more sulfa drugs may be included in the mediaat a concentration of from about 0.05 g/L to about 20 g/L, morepreferably of from about 0.1 g/L to about 10 g/L, and most preferablyfrom about 0.5 g/L to about 2.0 g/L. Such concentrations refer to thetotal concentration of all sulfa drugs in the composition.

The media of the invention may contain one or more surfactants as aselective agent. The surfactant may be a non-ionic surfactant, an ionicsurfactant, or an amphoteric surfactant. If an ionic surfactant, thesurfactant may be a cationic surfactant or an anionic surfactant.Preferred surfactants are anionic surfactants, such as aliphaticsulfates. The aliphatic sulfate may have a branched aliphatic chain or alinear aliphatic chain. Preferred aliphatic sulfates include7-ethyl-2-methyl-4-undecanol hydrogen sulfate or sodium salt thereof(Tergitol 4; CAS No. 139-88-8) and 7-ethyl-2-methyl-4-undecyl sulfate orsodium salt thereof (NIAPROOF® 4, available under Cat. No. N1404 fromSigma-Aldrich Co., St. Louis, Mo.). The 7-ethyl-2-methyl-4-undecylsulfate or a sodium salt thereof is particularly preferred. Thesealiphatic sulfates inhibit growth of Proteus spp. The one or moresurfactants may be included in the media at a concentration of fromabout 0.1 g/L to about 45 g/L, preferably of from about 0.2 g/L to about22.5 g/L, and more preferably of from about 1.0 g/L to about 4.5 g/L.

The media of the invention may contain one or more aminocoumarins as aselective agent. Aminocoumarins include clorobiocin, coumermycin A1, andnovobiocin. Novobiocin is a preferred aminocoumarin for inclusion in themedia of the invention. Novobiocin is a Gram-positive antibacterial.Novobiocin appears to facilitate Salmonella spp. recovery in selectiveenrichment media, probably by inhibiting the growth of competitivemicroorganisms. The one or more aminocoumarins may be included in themedia at a concentration of from about 0.002 g/L to about 1 g/L,preferably about 0.004 g/L to about 0.5 g/L, and more preferably about0.02 g/L to about 0.10 g/L.

The media of the invention may contain cycloheximide as a selectiveagent. Cycloheximide is an inhibitor of protein biosynthesis ineukaryotic organisms and thereby inhibits the growth of mold and yeast.Addition of cycloheximide is useful, as some yeasts can ferment2-deoxy-D-ribose. The cycloheximide may be included in the media at aconcentration of from about 0.001 g/L to about 1.0 g/L, preferably offrom about 0.002 g/L to about 0.5 g/L, and more preferably of from about0.01 g/L to about 0.10 g/L.

The media of the invention may contain a supravital stain as a selectiveagent. As used herein, “supravital stain” refers to a stain that entersand stains living cells, such as bacteria. Such stains are toxic tocertain organisms over time, some more so than others. Examples ofsupravital stains include gentian violet, crystal violet, brilliantgreen, bismark brown, safranin, methylene blue, and malachite blue,among others. Preferred supravital stains include those that are morehighly toxic to non-Salmonella microorganisms and/or non-Shigatoxin-producing E. coli microorganisms than Salmonella and/or non-Shigatoxin-producing E. coli. Brilliant green is a preferred supravital stainfor including the media. Brilliant green is a trimethylaryl dye thatinhibits certain non-Salmonella Gram-negative and Gram-positivebacteria. Brilliant green inhibits many commensal E. coli butsurprisingly has no effect against Shiga toxin-producing E. coli atconcentrations of about 1 mg/L. Brilliant green bleaches at low pH andtherefore does not interfere with the pH indicator reaction. Othersupravital stains, such as malachite blue, do not bleach effectively atlow pH and would therefore preclude the use of a chromogenic pHindicator. Such supravital stains, however, may be used when anindication of a change in pH is not desired or needed. The supravitalstain may be included in the media at a concentration of from about0.0001 g/L to about 0.5 g/L, preferably of from about 0.0002 g/L toabout 0.25 g/L, and more preferably of from about 0.001 g/L to about0.05 g/L.

The media of the invention may contain ascorbic acid as a selectiveagent. Ascorbic acid has inhibitory activity against some species ofCitrobacter. See U.S. Pat. No. 4,279,995 to Woods et al. Ascorbic acidmay be included in the media at a concentration of from about 0.05 g/Lto about 20 g/L, preferably of from about 0.1 g/L to about 10 g/L, andmore preferably of from about 0.5 g/L to about 2.0 g/L.

The media of the invention may contain bromobenzoic acid as a selectiveagent.

Bromobenzoic acid has inhibitory activity against some species ofCitrobacter. See U.S. Pat. No. 4,279,995 to Woods et al. Bromobenzoicacid may be included in the media at a concentration of from about 0.001g/L to about 1.0 g/L, preferably of from about 0.002 g/L to about 0.50g/L, and more preferably of from about 0.01 g/L to about 0.10 g/L. Themedia of the invention may contain myricetin as a selective agent.Myricetin inhibits Enterobacter and Klebsiella spp. Brilliant greeninhibits many commensal E. coli but surprisingly has no effect againstShiga toxin-producing E. coli or Salmonella at concentrations of about 1mg/L. Myricetin may be included in the media at a concentration of fromabout 0.001 g/L to about 1.0 g/L, preferably of from about 0.002 g/L toabout 0.50 g/L, and more preferably of from about 0.01 g/L to about 0.10g/L. Lower concentrations may be preferred for selection of Shigatoxin-producing E. coli.

The media of the invention may contain nitrofurantoin as a selectiveagent.

Nitrofurantoin is1-[[(5-Nitro-2-furanyl)methylene]amino]-2,4-imidazolidinedione and isavailable under Cat. No. N7878 from Sigma-Aldrich Co., St. Louis, Mo.Nitrofurantoin is typically used to treat urinary tract infection, andis often used against E. coli. As shown in the following examples,nitrofurantoin is surprisingly ineffective against Shiga toxin-producingE. coli and Salmonella and can therefore be used to select for thesemicroorganisms. The nitrofurantoin may be included in the media at aconcentration of from about 0.0001 g/L to about 0.1 g/L, preferably offrom about 0.0005 g/L to about 0.05 g/L, and more preferably of fromabout 0.001 g/L to about 0.01 g/L.

The media of the invention may contain one or more rifamycins as aselective agent.

Suitable rifamycins include rifamycins A, B, C, D, E, S, and SV as wellas the rifamycin derivatives rifampicin (or rifampin), rifabutin,rifapentine, and rifalazil. Rifampicin is preferred. The rifamycin maybe included in the media at a concentration of from about 0.0001 g/L toabout 0.2 g/L, preferably of from 0.0002 g/L to about 0.1 g/L, and morepreferably of from 0.001 g/L to about 0.02 g/L.

The media of the invention may contain one or more polyketides as aselective agent. Suitable polyketides include macrolide antibiotics suchas pikromycin, erythromycin A, clarithromycin, and azithromycin; polyeneantibiotics such as amphotericin; tetracycline; and doxacycline.Tetracycline and/or doxycycline are preferred. The polyketide may beincluded in the media at a concentration of from about 0.0001 g/L toabout 0.2 g/L, preferably of from 0.0002 g/L to about 0.1 g/L, and morepreferably of from 0.001 g/L to about 0.02 g/L.

The media of the invention may contain one or more oxazolidinones as aselective agent. Suitable oxazolidinones include linezolid (ZYVOX®,Pfizer, Inc., New York, N.Y.), posizolid, torezolid, radezolid(RX-1741), and cycloserine. Linezolid is preferred. The oxazolidinonemay be included in the media at a concentration of from about 0.0001 g/Lto about 0.2 g/L, preferably of from 0.0002 g/L to about 0.1 g/L, andmore preferably of from 0.001 g/L to about 0.02 g/L.

Some media of the invention comprise only one of a sulfa drug, asurfactant, an aminocoumarin, cycloheximide, a supravital stain,ascorbic acid, bromobenzoic acid, myricetin, nitrofurantoin, arifamycin, a polyketide, or an oxazolidinone as a selective agent. Somemedia of the invention comprise a sulfa drug in combination with anyone, all, or subcombinations of a surfactant, an aminocoumarin,cycloheximide, a supravital stain, ascorbic acid, bromobenzoic acid,myricetin, nitrofurantoin, a rifamycin, a polyketide, and anoxazolidinone as a selective agent. Some media of the invention comprisea surfactant in combination with any one, all, or subcombinations of asulfa drug, an aminocoumarin, cycloheximide, a supravital stain,ascorbic acid, bromobenzoic acid, myricetin, nitrofurantoin, arifamycin, a polyketide, and an oxazolidinone as a selective agent. Somemedia of the invention comprise an aminocoumarin in combination with anyone, all, or subcombinations of a sulfa drug, a surfactant,cycloheximide, a supravital stain, ascorbic acid, bromobenzoic acid,myricetin, nitrofurantoin, a rifamycin, a polyketide, and anoxazolidinone as a selective agent. Some media of the invention comprisecycloheximide in combination with any one, all, or subcombinations of asulfa drug, a surfactant, an aminocoumarin, a supravital stain, ascorbicacid, bromobenzoic acid, myricetin, nitrofurantoin, a rifamycin, apolyketide, and an oxazolidinone as a selective agent. Some media of theinvention comprise a supravital stain in combination with any one, all,or subcombinations of a sulfa drug, a surfactant, an aminocoumarin,cycloheximide, ascorbic acid, bromobenzoic acid, myricetin,nitrofurantoin, a rifamycin, a polyketide, and an oxazolidinone as aselective agent. Some media of the invention comprise ascorbic acid incombination with any one, all, or subcombinations of a sulfa drug, asurfactant, an aminocoumarin, cycloheximide, a supravital stain,bromobenzoic acid, myricetin, nitrofurantoin, a rifamycin, a polyketide,and an oxazolidinone as a selective agent. Some media of the inventioncomprise bromobenzoic acid in combination with any one, all, orsubcombinations of a sulfa drug, a surfactant, an aminocoumarin,cycloheximide, a supravital stain, ascorbic acid, myricetin,nitrofurantoin, a rifamycin, a polyketide, and an oxazolidinone as aselective agent. Some media of the invention comprise myricetin incombination with any one, all, or subcombinations of a sulfa drug, asurfactant, an aminocoumarin, cycloheximide, a supravital stain,ascorbic acid, bromobenzoic acid, nitrofurantoin, a rifamycin, apolyketide, and an oxazolidinone as a selective agent. Some media of theinvention comprise nitrofurantoin in combination with any one, all, orsubcombinations of a sulfa drug, a surfactant, an aminocoumarin,cycloheximide, a supravital stain, ascorbic acid, bromobenzoic acid,myricetin, a rifamycin, a polyketide, and an oxazolidinone as aselective agent. Some media of the invention comprise a rifamycin incombination with any one, all, or subcombinations of a sulfa drug, asurfactant, an aminocoumarin, cycloheximide, a supravital stain,ascorbic acid, bromobenzoic acid, myricetin, nitrofurantoin, apolyketide, and an oxazolidinone as a selective agent. Some media of theinvention comprise a polyketide in combination with any one, all, orsubcombinations of a sulfa drug, a surfactant, an aminocoumarin,cycloheximide, a supravital stain, ascorbic acid, bromobenzoic acid,myricetin, nitrofurantoin, a rifamycin, and an oxazolidinone as aselective agent. Some media of the invention comprise an oxazolidinonein combination with any one, all, or subcombinations of a sulfa drug, asurfactant, an aminocoumarin, cycloheximide, a supravital stain,ascorbic acid, bromobenzoic acid, myricetin, nitrofurantoin, arifamycin, and a polyketide as a selective agent.

The media of the invention may also contain one or more efflux pumpinhibitors (EPIs). As used herein, “efflux pump inhibitor” refers to anyagent capable of inhibiting a bacterial efflux pump. The EPI preferablyincreases the toxicity of selective agents in non-Salmonella andnon-Shiga toxin-producing E. coli microorganisms.

Phylogenetically, bacterial antibiotic efflux pumps belong to fivesuperfamilies (see reviews (Li X Z, Nikaido H. Efflux-mediated drugresistance in bacteria. Drugs 2004; 64:159-204) (Paulsen I T. Multidrugefflux pumps and resistance: regulation and evolution. Curr OpinMicrobiol 2003; 6:446-51) (Saier M H Jr. Tracing pathways of transportprotein evolution. Mol Microbiol 2003; 48:1145-56)), namely: (i) ABC(ATP-binding cassette), which are primary active transporters energizedby ATP hydrolysis; (ii) SMR [small multidrug resistance subfamily of theDMT (drug/metabolite transporters) superfamily]; (iii) MATE[multi-antimicrobial extrusion subfamily of the MOP(multidrug/oligosaccharidyl-lipid/polysaccharide flippases)superfamily]; (iv) MFS (major facilitator superfamily); and (v) RND(resistance/nodulation/division superfamily), which are all secondaryactive transporters driven by ion gradients. The MFS and RND pumps arethe most abundant. The MFS pumps are found in both Gram-positive andGram-negative bacteria, and are characterized by a relative narrowspectrum, recognizing usually one or sometimes a few antibiotic classes;the RND pumps are found exclusively in Gram-negative bacteria anddisplay an extremely wide spectrum of substrates (poly-selectivity),including not only several classes of antibiotics, but also antisepticcompounds, dyes, or detergents. See: (1) Levy S B. Active efflux, acommon mechanism for biocide and antibiotic resistance. J Appl Microbiol2002; 92 Suppl:65-71; (2) Li X Z, Nikaido H. Efflux-mediated drugresistance in bacteria. Drugs 2004; 64:159-204; (3) Lomovskaya O, TotrovM. Vacuuming the periplasm. J. Bacteriol 2005; 187:1879-83; (4) Poole K.Efflux-mediated antimicrobial resistance. J Antimicrob Chemother 2005;56:20-51; (5) Van Bambeke F, Glupczynski Y, Plesiat P, et al. Antibioticefflux pumps in prokaryotic cells: occurrence, impact on resistance andstrategies for the future of antimicrobial therapy. J AntimicrobChemother 2003; 51:1055-65; (6) Koronakis V. TolC—the bacterial exitduct for proteins and drugs. FEBS Lett 2003; 555:66-71; and (7) PiddockL J. Clinically relevant chromosomally encoded multidrug resistanceefflux pumps in bacteria. Clin Microbiol Rev 2006; 19:382-402.

Suitable EPIs that may be included in the media of the invention includephenothiazines (see Molnar J, Hever A, Fakla I, et al. Inhibition of thetransport function of membrane proteins by some substitutedphenothiazines in E. coli and multidrug resistant tumor cells.Anticancer Res 1997; 17:481-6), phenylpiperidines (see Kaatz G W,Moudgal V V, Seo S M, et al. Phenylpiperidine selective serotoninreuptake inhibitors interfere with multidrug efflux pump activity inStaphylococcus aureus. Int J Antimicrob Agents 2003; 22:254-61),tetracycline analogs (Nelson M L, Park B H, Andrews J S, et al.Inhibition of the tetracycline efflux antiport protein by13-thio-substituted 5-hydroxy-6-deoxytetracyclines. J Med Chem 1993;36:370-7; and Nelson M L, Park B H, Levy S B. Molecular requirements forthe inhibition of the tetracycline antiport protein and the effect ofpotent inhibitors on the growth of tetracycline-resistant bacteria. JMed Chem 1994; 37:1355-61), aminoglycoside analogs (Françoise VanBambeke, Jean-Marie Pages and Ving J. Lee. Inhibitors of BacterialEfflux Pumps as Adjuvants in Antibiotic Treatments and Diagnostic Toolsfor Detection of Resistance by Efflux. Recent Patents on Anti-InfectiveDrug Discovery, 2006, 1, 157-175), fluoroquinolone analogs (FrançoiseVan Bambeke, Jean-Marie Pages and Ving J. Lee. Inhibitors of BacterialEfflux Pumps as Adjuvants in Antibiotic Treatments and Diagnostic Toolsfor Detection of Resistance by Efflux. Recent Patents on Anti-InfectiveDrug Discovery, 2006, 1, 157-175), quinoline derivatives (Mahamoud A,Chevalier J, Davin-Regli A, et al. Quinolone derivatives as promisinginhibitors of antibiotic efflux pump in multidrug resistant Enterobacteraerogenes. Curr Drug Targets 2006; 7:843-7), peptidomimetics (LomovskayaO, Bostian K A. Practical applications and feasibility of efflux pumpinhibitors in the clinic—a vision for applied use. Biochem Pharmacol2006; 71:910-18), pyridopyrimidines (Nakayama K, Ishida Y, Ohtsuka M, etal. MexAB-OprM-specific efflux pump inhibitors in Pseudomonasaeruginosa. Part 1: discovery and early strategies for leadoptimization. Bioorg Med Chem Lett 2003; 13:4201-4; Nakayama K, IshidaY, Ohtsuka M, et al. MexAB-OprM specific efflux pump inhibitors inPseudomonas aeruginosa. Part 2: achieving activity in vivo through theuse of alternative scaffolds. Bioorg Med Chem Lett 2003; 13:4205-8;Nakayama K, Kawato H, Watanabe J, et al. MexAB-OprM specific efflux pumpinhibitors in Pseudomonas aeruginosa. Part 3: Optimization of potency inthe pyridopyrimidine series through the application of a pharmacophoremodel. Bioorg Med Chem Lett 2004; 14:475-9; Nakayama K, Kuru N, OhtsukaM, et al. MexAB-OprM specific efflux pump inhibitors in Pseudomonasaeruginosa. Part 4: Addressing the problem of poor stability due tophotoisomerization of an acrylic acid moiety. Bioorg Med Chem Lett 2004;14:2493-7; and Yoshida K, Nakayama K, Kuru N, et al. MexAB-OprM specificefflux pump inhibitors in Pseudomonas aeruginosa. Part 5:Carbon-substituted analogues at the C-2 position. Bioorg Med Chem 2006;14:1993-2004), arylpiperidines (Thorarensen A, Presley-Bodnar A L,Marotti K R, et al. 3-Arylpiperidines as potentiators of existingantibacterial agents. Bioorg Med Chem Lett 2001; 11:1903-6), andarylpiperazines (Bohnert J A, Kern W V. Selected arylpiperazines arecapable of reversing multidrug resistance in Escherichia colioverexpressing RND efflux pumps. Antimicrob Agents Chemother 2005;49:849-52; Schumacher A, Steinke P, Bohnert J A, et al. Effect of1-(1-naphthylmethyl)-piperazine, a novel putative efflux pump inhibitor,on antimicrobial drug susceptibility in clinical isolates ofEnterobacteriaceae other than Escherichia coli. J Antimicrob Chemother2006; 57:344-8; Kern W V, Steinke P, Schumacher A, et al. Effect of1-(1-naphthylmethyl)-piperazine, a novel putative efflux pump inhibitor,on antimicrobial drug susceptibility in clinical isolates of Escherichiacoli. J Antimicrob Chemother 2006; 57:339-43; Pannek S, Higgins P G,Steinke P, et al. Multidrug efflux inhibition in Acinetobacterbaumannii: comparison between 1-(1-naphthylmethyl)-piperazine andphenyl-arginine-beta-naphthylamide. J Antimicrob Chemother 2006;57:970-4). See also Mahamoud, A. et al. Antibiotic efflux pumps inGram-negative bacteria: the inhibitor response strategy. J. Antimicrob.Chemoth. 59(6):1223-1229. 2007.

Suitable phenothiazines include promethazine and 3,7,8,-trihydroxy-,7,8-dihydroxy, 7,8-diacetoxy-, 7,8dimetoxy-, 7-semicarbazone-, and5-oxo-chlorpromazine derivatives, among others. Suitablephenylpiperidines include the paroxetine isomer NNC 20-7052, amongothers. Suitable tetracycline analogs include 13-(alkylthio) and13-(arylthio) derivatives of 5-hydroxy-6-deoxytetracycline, amongothers. Suitable aminoglycoside analogs include the aminoglycosidesdescribed in U.S. Pat. No. 7,829,543, among others. Suitablefluoroquinolone analogs include those described in WO0209758A2 andWO0209758A3, among others. Suitable quinoline derivatives includealkylamino-, alkyl-alkoxy-, thioalkoxy-, and halo-quinoline derivatives(e.g., chloroquinoline derivatives) and those having piperidinoethylchains, among others. A preferred quinoline derivative is4-chloroquinoline. Suitable peptidomimetics include MC-207 110 orphenylalanine arginyl β-naphthylamide (PAβN), and derivatives thereof,among others. Suitable arylpiperidines include 3-arylpiperidinederivatives, among others. Suitable arylpiperazines includearylpiperazines, including 1-(1-naphthylmethyl)piperazine and others.

The EPIs are preferably selected from the group consisting ofarylpiperazines, such as 1-(1-naphthylmethyl)piperazine (NMP; CAS No.40675-81-8; available as Cat. No. 651699, Sigma-Aldrich Co., St. Louis,Mo.), and quinoline derivatives, such as 4-chloroquinoline (4-CQ; CASNo. 611-35-8; available as Cat. No. C70509, Sigma-Aldrich Co., St.Louis, Mo.). The NMP and 4-CQ may be included individually or together.

As shown in the following Examples, combinations of an EPI withpolyketide, rifamycin, or oxazolidinone antibiotics can increase theactivity of these antibiotics against Enterobacteriacae and othermicroorganisms without substantially affecting growth and fermentationof Salmonella species and/or Shiga toxin-producing E. coli strains.Accordingly, the one or more selective agents and the efflux pumpinhibitor are present in the media of the invention in amounts effectiveto inhibit growth of at least one non-Salmonella species and/or at leastone non-Shiga toxin-producing E. coli strain to a greater extent thanSalmonella species (such as Salmonella enterica) and/or Shigatoxin-producing E. coli strains. It is preferred that the combination ofthe one or more selective agents and the efflux pump inhibitor arepresent in an amount that does not substantially affect the growth ormetabolism of Salmonella species (such as Salmonella enterica) and/orShiga toxin-producing E. coli strains.

The media described herein can be provided in a hydrated form, such asin the form of a liquid or gel-like (e.g., agar) medium, or in a driedform. If in a dried form, the components are preferably present in aproportion such that addition of water or other solvents provides eachof the components within the concentration ranges described above. Inaddition, the media, whether in died or hydrated form, may be providedin separate combinations, e.g., basal media and one or more supplements,as described in the following examples.

Methods of using the media of the invention are apparent from thefollowing examples. The media of the invention can be used to select forSalmonella and/or Shiga toxin-producing E. coli from othermicroorganisms, the latter including Citrobacter spp. (e.g., Citrobacterfreundii, Citrobacter koseri, etc.), non-Shiga toxin-producing E. coli,and/or commensal E. coli generally. The media of the invention can beused for detecting Salmonella and/or Shiga toxin-producing E. coli.Specifically, the media of the invention can be used for cultivating orselecting for Salmonella (including Salmonella enterica); cultivating orselecting for Shiga toxin-producing E. coli (including E. coli having atype selected from O157, O145, O104, O26, O111, O103, and O91); orco-cultivating or co-selecting for Salmonella (including Salmonellaenterica) and Shiga toxin-producing E. coli (including E. coli having atype selected from O157, O145, O104, O26, O111, O103, and O91).

Kits for use of the media of the invention may include any combinationof components described herein, such as in the following examples.

Concentrations other than those explicitly described herein, such asabove and below the stated ranges, are included in the invention.

The elements and method steps described herein can be used in anycombination whether explicitly described or not.

All combinations of method steps described herein can be performed inany order, unless otherwise specified or clearly implied to the contraryby the context in which the referenced combination is made.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the content clearly dictates otherwise.

Numerical ranges as used herein are intended to include every number andsubset of numbers contained within that range, whether specificallydisclosed or not. Further, these numerical ranges should be construed asproviding support for a claim directed to any number or subset ofnumbers in that range. For example, a disclosure of from 1 to 10 shouldbe construed as supporting a range of from 2 to 8, from 3 to 7, from 5to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

All patents, patent publications, and peer-reviewed publications (i.e.,“references”) cited herein are expressly incorporated by reference tothe same extent as if each individual reference were specifically andindividually indicated as being incorporated by reference. In case ofconflict between the present disclosure and the incorporated references,the present disclosure controls.

It is understood that the invention is not confined to the particularconstruction and arrangement of parts herein illustrated and described,but embraces such modified forms thereof as come within the scope of thefollowing claims.

EXAMPLES

The following working examples are intended to illustrate the featuresof the present invention without limiting the particular components orpotential use of the media.

Example 1: Salmonella Indicator Broth-1 (SIB-1)

Several experiments were conducted with an EPI-supplemented medium,referred to herein as “SIB-1.”

Example 1.1: Summary

The Salmonella Indicator Broth-1, (SIB-1), is a single step selectiveenrichment indicator broth to be used as simple screening test for thepresence of Salmonella spp. in environmental samples. This test permitsthe end user to avoid the multi-step process of sample processing toidentify presumptively positive samples as exemplified by standard U.S.reference methods. SIB-1 permits the outgrowth of Salmonella whileinhibiting the growth of competitive gram-negative and gram-positivemicro-flora. Growth of Salmonella-positive cultures results in a visualcolor change of the medium from purple to yellow when the sample isgrown at 37±1° C.

Performance of SIB-1 was evaluated in five different categories:inclusivity-exclusivity, methods comparison, ruggedness, lot-to-lotvariability, and shelf stability.

The inclusivity panel included 100 different Salmonella serovars ofwhich 98 were positive for SIB-1 during the 30 to 48 hour incubationperiod.

The exclusivity panel included 33 different non-Salmonellamicroorganisms of which 31 were SIB-1 negative during the incubationperiod.

Methods comparison studies included four different surfaces, S. Newporton plastic, S. Anatum on sealed concrete, S. Abaetetuba on ceramic tileand S. Typhimurium in the presence of one log excess of C. freundii.Results of the methods comparison studies demonstrated no statisticaldifference between the SIB-1 method and the FDA-BAM reference method, asmeasured by the Mantel-Haenszel chi-square test. The overall sensitivityrelative to the reference method across all four surfaces was >100% (itis possible bacterial growth occurred during the study period). Therewere no significant differences between SIB-1 and the reference methodon any of the surfaces tested.

Ruggedness studies demonstrated little variation in test results whenSIB-1 incubation temperatures were varied over a six degree centigraderange, 34° C. to 40° C. Lot to lot consistency results suggests nodetectable differences in manufactured goods using two referenceSalmonella serovars and one non-Salmonella microorganism.

Example 1.2: Definitions

Relative Sensitivity: Defined as the number of samples testing positiveby the SIB-1 method divided by the number of samples testing positive bythe reference culture procedure.

Statistical Data Analysis: The Mantel-Haenszel chi-square formula forunmatched test portions was used for the statistical analysis (1). AChi-square value <3.84 indicates that the proportions positive for thealternative and the reference method are not statistically different atthe 5% level of significance. This criterion must be satisfied for eachlevel of each surface type. However, a significant difference betweenthe proportions positive for the two methods is acceptable provided thatthe alternative method demonstrates superior recovery to the referencemethod.

Example 1.3: Principle

The principle of SIB-1 utilizes two operating conditions, the firstselective enrichment of the Salmonella population from the backgroundmicroflora and secondly the simultaneous metabolism of a very specificSalmonella substrate. SIB-1 is a balanced blend of proprietary selectiveagents highly restrictive to non-Salmonella bacteria and combining ahighly specific metabolic substrate for Salmonella. As the selectedpopulation grows out the media becomes acidified and an incorporated pHindicator detects the pH change by a color shift from purple to yellow.

Example 1.4: Preparation of Basal SIB Medium

Peptone 10 g/L, MgCl₂ hexahydrate 13 g/L, bromocresol purple 0.02 g/L,and sulfanilamide 0.9 g/L were dissolved in 950 mL de-ionized, distilledwater; autoclaved under standard sterilization cycle conditions, 121° C.at 3 atm for 15 minutes; and cooled to about 30° C. The followingcomponents were then added as filter-sterilized supplements:sulfathiazole 0.1 g/L; NIAPROOF® 4 1.5 g/L; 2-deoxy-D-ribose 5 g/L;cycloheximide 0.05 g/L; ascorbic acid 1 g/L; p-bromobenzoic acid 0.04g/L; novobiocin 0.02 g/L; brilliant green 0.001 g/L; and myricetin 0.03g/L.

Example 1.5: Preparation of SIB-1

Basal SIB was supplemented with 1-(1-naphthylmethyl)piperazine (NMP) ata final concentration of 0.2 mM (0.044 g/L) to generate SIB-1.

Example 1.6: SIB-1 Kit Components

Kits for methods employing Salmonella selection and detection include15-mL screw cap vials containing SIB-1. The kits may also include 3M™ENVIRO SWAB environmental sample collecting swabs, available through 3MCompany (St. Paul, Minn.) or Paradigm Diagnostics, Inc. (St. Paul,Minn.), or equivalent. Alternatives include WHIRL-PAK® sampling bags(Nasco Industries, Inc., Fort Atkinson, Wis.) with pre-moistened carcasssponges, or equivalent.

Example 1.7: Incubations

Incubations were conducted in a temperature-thermostated incubator(preferred) or heating block (32° C. to 40° C.).

Example 1.8: Standard Reference Materials

Reference strains were obtained through the following sources,Microbiologics, Inc., St. Cloud, Minn., National Collection of TypeCultures (NCTC) Health Protection Agency Culture Collection, PortonDown, UK., Prof. Francisco Diez-Gonzalez, Department of Food Science,University of Minnesota, St. Paul, Minn., and the American type CultureCollection (ATCC), Bethesda, Md. Sero-groups were identified throughreference to the handbook of Salmonella antigenic structures (Grimont,Patrick A. D. & Weill, Francois-Xavier. ANTIGENIC FORMULAE OF THESALMONELLA SEROVARS. 2007, 9th edition).

Example 1.9: General Sample Preparation

Pure cultures were obtained from Health Protection Agency, NCTC, PortonDown, U K and Microbiologics, Inc., St. Cloud, Minn., the University ofPennsylvania School of Veterinary Medicine, Center for ReferenceSalmonella and Microbiology Labs of Dr. Diez and Dr. Feirtag at theUniversity of Minnesota, St. Paul, Minn. Original pure cultures weregrown overnight in sterile Tryptic Soy Broth (TSB) or Brain HeartInfusion broth (BHI) at 32-37° C. In order to have reproduciblecultures, 50% glycerol stock solutions of cultures were prepared andstored in a freezer. Fifty percent glycerol stock cultures were preparedby diluting 500 μL of pure culture grown overnight with 500 μL sterileglycerol. Then 200 μL portions were filled into sterile 2 mL centrifugetubes, capped and kept in a freezer at −15° C. until the day before use.On the day before use, a loop-full of freezer-stored stock cultures wereinoculated into 5 mL of sterile TSB, the same loop was streaked ontotryptic soy agar (TSA). The plates were incubated overnight at 37±1° C.TSA plates were checked for visible contamination, based on colonymorphology. If the TSA plates suggested there was no visiblecontamination, logarithmic level dilutions were made into sterilepeptone solutions (0.1% peptone water). Aliquots containing 100 μL of 6and 7 log dilutions from overnight cultures were plated onto TSA forestimating the cell concentration (CFU/mL) and checked forcontamination.

Dilutions of pure cultures aforementioned were used forinclusivity-exclusivity, method comparison, ruggedness and lot to-lotvariability, and shelf stability studies.

Example 1.10: Environmental Sample Preparation and Analysis

Environmental samples were taken with sampling devices following U.S.FDA Bacteriological Analytical Manual (BAM) recommendations for sampling(U.S. Food and Drug Administration (2011) Bacteriological AnalyticalManual, Chapter 5). The sampling devices were returned back into theoriginal sterile container. One unit (15 mL) of SIB-1 media wasaseptically added to each sterile container to fully submerge theapplicator tip and was incubated in an upright position for 30 to 48hours at 37±1° C. The color of the media was checked. The sample wascalled presumptive positive for Salmonella if the color of the mediachanged from purple color to a yellow color.

In order to confirm the negatives, purple/pale colorless-colored(presumptive negative) tubes were incubated for a total of 48 hours. Atleast one negative control was run in each set of analysis. A negativecontrol was an unused sampling device containing one unit of SIB-1,incubated alongside the samples.

Example 1.11: Inclusivity-Exclusivity Studies

Serial dilutions from overnight grown pure cultures of 100 differentSalmonella and 33 different non-Salmonella cultures were made intosterile 0.1% peptone water. One hundred microliters from log −8 dilution(˜1.0E+3 CFU/mL) were plated onto TSA plates for estimating the numberof cells tested. For the inclusivity studies 0.1 mL (estimated cellamount ranging from 10 to 100 CFU of Salmonella) of a dilution aliquotwas aseptically transferred onto the top of the sampling sponge. For theexclusivity studies 0.2 mL (estimated cell amount >1,000,000 CFU ofnon-Salmonella) of a dilution aliquot was aseptically transferred ontothe top of the sampling sponge. The sampling device was placed back intothe sterile tube and submerged in 15 mL of SIB-1 media. The results aregiven in Tables 1-1 and 1-2 respectively.

TABLE 1-1 Results of Inclusivity Test for SIB-1 SIB-1 Medium PresumptiveSero- Serovar Source Origin Color Result group S. Adelaide U of MN94679420 Meat meal Yellow + O S. Agona U of MN inv Soybean Yellow + B95650951 meal S. Albany U of MN 2009595 Frozen Yellow + C3 fish paste S.Anatum U of MN 95645854 Chicken Yellow + E1 feed S. Bovismorbificans Uof MN 3064124 Vietnam Yellow + C2 S. Carrau U of MN 2003413 FrozenYellow + H shrimp S. Cerro U of MN 94713965 Poultry Yellow + K feed S.Cubana U of MN 94679421 Swine Blue G2 feed S. Chester U of MN 3063650Frozen Yellow + B tilapia fish S. Emek U of MN 3063892 Frozen Yellow +C3 catfish S. Enteritidis U of MN 95657613 Ice cream Yellow + D1 S. GiveU of MN 1829352 Lobster Yellow + E1 tail S. Gloucester U of MN 1676771Sesame Yellow + B seeds S. Hvittingfoss U of MN 200373 Frozen Yellow + Ifrog legs S. Infantis U of MN 2015422 Frozen Yellow + C1 lobster tail S.Javiana U of MN 1842147 Frozen Yellow + D1 shrimp S. Kentucky U of MN95-690-012 Cottonseed Yellow + C3 S. Lille U of MN 95-713-959 ChickenYellow + C1 feed S. Mbandaka U of MN 95690014 Soybean Yellow + C1 mealS. Meleagridis U of MN 1949345 Frozen Yellow + E1 shrimp S. Montevideo Uof MN 95573493 Raw eggs Yellow + C1 S. Muenchen U of MN 1842204 FrozenYellow + C2 shrimp S. U of MN 1842304 Frozen Yellow + E1 Newbrunswickshrimp S. Nashua U of MN 2006036 Poultry Yellow + M feed S. Newport U ofMN 2006038 Frozen Yellow + C2 lobster tail S. Penilla U of MN 1949289Frozen Yellow + M shrimp S. Poona U of MN 1103174 White Yellow + G1pepper S. Sterrenbos U of MN 1842082 Frozen Yellow + C3 shrimp S.Thompson U of MN 95657618 Ice cream Yellow + C1 S. Weltevreden U of MN1950358 Dried ling Yellow + E1 shrimp S. Typhimurium U of MN 3019907Salted Yellow + B dune egg S. Worthington U of MN 95-713-958 ChickenYellow + G2 feed S. Kumasi U of MN 1929854 Frozen Yellow + N crab meatS. Rubislaw U of MN 2004976 Frozen Yellow + F shrimp S. Goodwood U of MNFaeces Yellow + E4 S. Senftenberg U of MN Sewage Yellow + E4 S. Ohio Uof MN Animal Yellow + C1 feed S. Limete U of MN Yellow + B S. TennesseeU of MN Soybean Yellow + C1 meal S. Newington U of MN Wild Yellow + Bpoultry S. Aberdeen NCTC 5791 Infantile Yellow + F diarrhea S.Aequatoria NCTC 7891 African Yellow + C1 zoonosis S. Alabama NCTC 9868Human Yellow + B faeces S. Altendorf NCTC 10546 Yellow + B S. AustinNCTC 8447 Yellow + C1 S. Ball NCTC 9870 Yellow + B S. Berkeley NCTC 8260Diseased Yellow + U turkey S. Brookfield NCTC 10946 Yellow + O66 S.California NCTC 6018 Animal Yellow + B feed S. Canastel NCTC 6948 AnimalYellow + D1 feed S. Carmel NCTC 9872 Infantile Yellow + O17 diarrhea S.Champaign NCTC 6851 Hen liver Yellow + Q S. Chicago NCTC 9873 Yellow + MS. Colombo NCTC 9922 Sheep Yellow + P S. Ealing NCTC 11949 DriedYellow + O baby milk S. Dahlem NCTC 9949 Cattle Yellow + Y S. GallinarumNCTC 10532 Poultry Blue − D1 S. Houten NCTC 10401 Reptile Yellow + O43S. Kottbus NCTC 5753 Faeces Yellow + C2 S. Illinois NCTC 8498 PoultsYellow + E3 S. Lexington NCTC 6244 Soybean Yellow + E1 S. ManchesterNCTC 7372 Yellow + C2 S. Minnesota NCTC 5800 Swine Yellow + L S.Mississippi NCTC 6487 Faeces Yellow + G2 S. Napoli NCTC 6853 FoodYellow + D1 handlers S. Pensacola NCTC 6946 Yellow + D1 S. Pretoria NCTC6234 Meat Yellow + F S. Shanghai NCTC 9791 Yellow + I S. Sunsvall NCTC9787 Dried egg Yellow + H S. Waycross NCTC 7401 Urine Yellow + S S.Alachua U Penn STS 6 Swine Yellow + O S. Choleraesuis ATCC 10708 FishYellow + C S. Arkansas U Penn STS 11 Yellow + B S. Blockley U Penn STS15 Environment Yellow + C2 S. Brandenburg U Penn STS 18 Swine Yellow + BS. Derby U Penn STS 22 Polluted Yellow + B water S. Dublin U Penn STS 27Cattle Yellow + D1 S. Hadar U Penn STS 45 Turkey Yellow + C2 S.Heidelberg U Penn STS 48 Poultry Yellow + B S. London U Penn STS 64Polluted Yellow + E1 water S. Manhattan U Penn STS 65 Avian Yellow + C2S. Oranienburg U Penn STS 83 Egg Yellow + C1 S. Panama U Penn STS 86Infantile Yellow + D1 diarrhea S. Paratyphis ATCC 13314 Sewage Yellow +A S. Saint Paul U of MN Milk Yellow + B powder S. Schwarzengrund U PennSTS 95 Chicken Yellow + B S. Stanley U Penn STS100 Reptile Yellow + B S.Urbana U Penn STS110 Reptile Yellow + N S. Johannesburg U Penn STS 56Meat meal Yellow + R S. Thomasville U Penn STS103 Poultry Yellow + E3meal S. Virchow U Penn STS 112 Basil Yellow + C1 S. Abaetetuba ATCC35640 Fresh Yellow + F water S. Choleraesuis ATCC 12011 Swine Yellow + Bvar. Kunzendorf S. Vallore ATCC 15611 Yellow + B S. Paratyphis U of MN2014696 Frozen Yellow + B frog legs S. Tallahassee ATCC 12002 Yellow +C3 S. Salford U of MN 2009532 Oregano Yellow + I turkey S. Birmingham Uof MN Alfalfa Yellow + E1 DI95764802 seed S. Brunei U of MN 1680318Frozen Yellow + C3 Shrimp S. Ikeja U of MN 3019543 Frozen Yellow + E1Shrimp S. Cubana UPenn Swine Yellow + G2

TABLE 1-2 Results of Exclusivity Test for SIB-1 Species Source OriginSIB-1 Result Klebsiella NCTC 9633 Sputum − pneumoniae Proteus mirabilisATCC 12453 GI tract − Citrobacter freundii NCTC 9750 Soil − Escherichiacoli ATCC 13706 GI tract − Escherichia coli ATCC 14948 GI tract − Hafniaalvei ATCC 700025 Brewery − fermentation samples Serratia liquefaciensATCC 27592 − Morganella morganii ATCC 25829 − subsp.morganii PseudomonasATCC 10145 − aeruginosa Providencia rettgeri ATCC 9250 − EnterobacterATCC 51816 − amnigenus Enterobacter ATCC 13048 − aerogenes Shigellasonnei ATCC 25931 − Shigella flexneri ATCC 9199 − Staphylococcus ATCC14990 − epidermidis Staphylococcus ATCC 700699 − aureus Serratiamarcescens ATCC 13880 Polenta − Enterobacter cloacae ATCC 23355 −subsp.cloacae Enterobacter ATCC 33028 − gergoviae Klebsiella oxytocaATCC 13182 − Providencia ATCC 16529 − wickerhamii Shigella boydii ATCC9207 − Staphylococcus NCTC 12973 − aureus Yersinia ATCC 23715 −enterocolitica subsp. enterocolitica Yersinia ruckerii ATCC 29473 −Citrobacter freundii ATCC 8090 + Citrobacter braakii ATCC 43162 −Citrobacter koseri ATCC 27156 + Escherichia coli NCIMB 11943 −Escherichia coli NCTC 10538 − Listeria ATCC 13932 − monocytogenesListeria innocua ATCC 33090 − Pasteurella ATCC 12945 − multocida subsp.multocida Providencia stuartii ATCC 33672 − Edwardsiella tarda ATCC15947 −

Example 1.12: Methods Comparison Studies

Method comparison studies were done for four different Salmonellaspecies paired with four different common food environmental surfaces;S. Abaetetuba on ceramic tiles, S. Anatum on sealed concrete, S. Newporton plastic surface, and S. Typhimurium in the presence of one log excessC. freundii on stainless steel.

Example 1.12.1: FDA-BAM Method

The FDA-BAM method calls for a multi-step procedure as described.Following the 2 hour holding period after sampling, 3M™ ENVIROSWABenvironmental sample collecting swabs were transferred to sterilestomacher bags and enriched with 225 mL of Lactose broth. The sampleswere allowed to stand for 60±5 minutes at room temperature and thenincubated for 24±2 hours at 35±1° C. No pH adjustment was necessaryprior to incubation. After incubation, 0.1 mL of primary enrichment foreach sample was transferred to 10 mL of Rappaport-Vasilliadis medium(RV) and 1.0 mL to 10 mL of Tetrathionate (TT) broth. The RV broth wasincubated at 42±1° C. for 24±2 hours and the TT broth was incubated at35±1° C. for 24±2 hours. Following incubation, a loopful (10 μL) of eachsecondary enrichment was streaked to bismuth sulfate (BS), xylose lysinedesoxycholate (XLD), and Hektoen enteric (HE) selective agars andincubated at 35±1° C. for 24±2 hours. The BS plates that were negativeat 24 hours were then re-incubated for an additional 24 hours at 35±1°C. A suspect colony from each selective agar was picked and stabbed toTriple Sugar Iron (TSI) agar and Lysine Iron Agar (LIA) plates andstreaked to a tryptic soy agar (TSA) plate. The TSI, LIA, and TSA plateswere incubated at 35±1° C. for 24±2 hours. Growth from each TSA platewas used to conduct the polyvalent somatic 0 serological test, MICROGEN™SALMONELLA LATEX test (Microgen Bioproducts Ltd., Surrey, UK) andbiochemical tests for confirmation. Final confirmations were conductedwith MICROGEN™ GN-ID biochemical panels (Microgen Bioproducts Ltd.,Surrey, UK).

Example 1.12.2: SIB-1 Method

Following the 2 hour holding period after sampling, excess DEneutralizing broth from the swab tubes (3M™ ENVIROSWAB) was removed and15 mL of Salmonella Indicator Broth was added to the swab tube. Thesamples were then incubated at 37±1° C. for 24-48 hours. After 24 hours,each sample was examined for presumptive positive results (broth colorchange from blue to yellow). Presumptive positives at 24 hours werestreaked to the reference agars for the specified reference method andre-incubated for an additional 24 hours. Positive and negative samples,yellow and purple respectively were streaked onto XLD, HE and BS andfollowed through to confirmation as described for the FDA-BAM method.

Example 1.12.3: S. Newport on Plastic Surface

Serial logarithmic dilutions of overnight culture of S. Newport in BHIwere made into sterile BHI. A one-hundred μL volume of log −7 and log −8dilutions was plated onto TSA plates for estimating the number of cellsloaded onto 4″×4″ zones on plastic. Polypropylene plastic cutting boardswere bought from a local department store. 4″×4″ zones were marked onthe surface of the cutting boards. They were wrapped in aluminum foiland autoclaved for 15 min at 120° C. Plastic cutting boards were keptwrapped until the time of inoculation. 1 The inoculation of surfaceswere done at 0.5 mL/4″×4″ surface from the log −7 (high) and log-8 (low)dilutions, corresponding approximately to 2.18 log CFU and 1.18 log CFUof S. Newport consecutively, twenty replicates for each level permethod. Inoculations on surfaces were spread within the corresponding4″×4″ zone with the help of a disposable sterile spreader. After thatsurfaces were left to dry min 18 hours at room temperature. After thesamples were dried on the surfaces for a minimum of 18 hours, they wereremoved by swabbing using 3M™ ENVIROSWAB environmental sample collectingswabs saturated with ten mL of DE broth. Both test method (SIB-1) andreference method (FDA-BAM) was done in twenty replicates per level (highand low) of inoculations. In order to confirm the presence of Salmonellain all test samples, all samples were streaked onto HE, XLD and BS agarafter at the end of 48th hour at 37±1° C. Dark green colonies grown onHE were selected and processed as described for GN-ID analysis andpolyvalent somatic 0 serological tests. Five un-inoculated samples wereassayed. The summary of the data for S. Newport on plastic is given inTable 2.

As seen from Table 2, SIB-1 was comparable to the reference method. Thereference method gave two more positives at the low level than the testmethod. Although the Chi Square values indicated that the test methodand the reference method were not significantly different at the lowlevel.

Example 1.12.4: S. Anatum on Sealed Concrete

S. Anatum stock culture grown overnight in BHI. Serial logarithmicdilutions of S. Anatum were made into sterile BHI. A one-hundred μLvolume of log −7 and log −8 dilutions were plated onto TSA plates forestimating the number of cells loaded onto 4″×4″ zones on sealedconcrete blocks. Concrete blocks (15.5″×7.5″×3.5″; length×width×depth)were purchased from a local HOME DEPOT® supply store. Blocks were sealedwith a solvent-based concrete sealer (BW Crete Seal 25 LV, St. Paul,Minn.). After a minimum of two coats of sealant was placed on theblocks, they were dried in a chemical hood until all the solvent hadevaporated (minimum of 24 hours). Zones of 4″×4″ were marked on thesealed side using a permanent marker. Before inoculations, sealedconcrete blocks were sprayed with ethanol and allowed to air dry. Theethanol was air dried for at least minutes but no more than an hour. Theinoculation of sealed concrete surfaces were done at 0.5 mL/surface fromthe log-7 S. Anatum (high) and log-8 S. Anatum (low) dilutions,corresponding approximately to 2.22 log CFU and 1.25 log CFUrespectively, twenty replicates for each level, per method. Inoculationson surfaces were spread within the corresponding 4″×4″ zone. Surfaceswere left to dry for at least 18 hours at room temperature.

After the samples were dried on the surfaces for a minimum of 18 hours,they were removed by swabbing using 3M™ ENVIROSWAB environmental samplecollecting swabs. Both test methods SIB-1 method and reference method(FDA-BAM) were done in twenty replicates per level (high and low) ofinoculations. Five un-inoculated samples were assayed.

Table 2 shows the reference method identified a single sample more thanthe test method at the low level. Accordingly chi square valuessuggested that there was no significant difference between the testmethod and the reference method at both of the levels tested.

Example 1.12.5: S. Abaetetuba on Ceramic Tile

Serial logarithmic dilutions of overnight culture of S. Abaetetuba inBHI were made into sterile BHI. Aliquots containing 100 μL of log −8 andlog −9 dilutions were plated onto TSA plates for estimating the numberof cells loaded onto 4″×4″ ceramic tiles. Ceramic tiles at 4×4 inches indimensions were bought from a local HOME DEPOT® supply store. They werewrapped in aluminum foil and autoclaved for 15 min at 121° C. All tileswere kept wrapped at room temperature until the time of inoculation.

The inoculation of surfaces was done at 0. 50 mL/4″×4″ surface from thelog −8 (high) and log-9 (low) dilutions, corresponding approximately to1.67 log CFU and 0.60 log CFU respectively, twenty replicates for eachlevel, per method. Inoculations on surfaces were spread within thecorresponding 4″×4″ zone with the help of sterile spreaders. Surfaceswere left to dry for at least 18 hours at room temperature. After thesamples were dried on the surfaces for a minimum of 18 hours, they wereremoved by swabbing using the 3M™ ENVIROSWAB swabs. Both the test method(SIB-1) and the reference method (FDA-BAM) were done in twentyreplicates per level (high and low) of inoculations. Five un-inoculatedsamples were assayed.

In order to confirm the presence-absence of Salmonella in all testsamples, all tubes of SIB-1 were streaked onto HE, XLD and BS plates atthe end of 48th hour of incubation. Dark green colonies with grown on HEwere processed as described for immunoassay and GN-ID analysis forconfirmation. Summary of the data for S. Abaetetuba on tile is given inTable 2.

As seen from the Table 2 below, SIB-1 was slightly more sensitive thanthe reference method. The reference method resulted in fewer positives 7versus 10, than the test method at the low level. Chi-square value alsoshowed that the two methods were not significantly different from eachother at the low level testing.

Example 1.12.6: Independent Laboratory Surface Comparison Study: S.Typhimurium: C. freundii on Stainless Steel

For the analysis of stainless steel surfaces, a total of 45 samples forboth the SIB-1 and FDA/BAM were analyzed for method comparison. Withineach sample set there were 20 low-level, 20 high-level, and 5un-inoculated samples. The target levels of S. Typhimurium ATCC #14028used for challenging the stainless steel surfaces were as follows: 1-50CFU/4″×4″ (100 cm²) surface area for the low-level samples, 50-100CFU/4″×4″ (100 cm²) surface area for the high-level samples, and 0CFU/4″×4″ (100 cm²) surface area for the un-inoculated control samples.Additionally, the surfaces were inoculated with C. freundii ATCC #8090at 10 times the level of the S. Typhimurium to simulate the performanceof the target organism in the presence of a competing microflora. Theinocula were prepared in Brain Heart Infusion Broth (BHI) incubated for24±2 hours at 37±1° C. Following incubation, the broth inocula wereserial diluted using BHI until the target inoculum level was reached.The stainless steel surfaces were inoculated with 0.25 mL from both theS. Typhimurium and C. freundii inocula and then allowed to dry atambient temperature for 16-24 hours.

Results obtained from the stainless steel environmental surfaces assayedby the SIB-1 method were comparable to those analyzed by the FDA/BAMreference method for the detection of Salmonella. See Table 1-3 AMantel-Haenszel chi-square analysis for unmatched test portions betweenthe SIB-1 method and the reference method produced a value of 2.05 forthe low-level test portions and a value of 0.61 for the high-level testportions.

The values obtained for the matrix indicate that there was nostatistically significant difference between the number of confirmedpositive results obtained by the two methods being compared at bothlevels, 5 positives for the SIB-1 method compared to 3 positives for theFDA-BAM procedure.

TABLE 1-3 Summary of Method Comparison Studies of SIB at 48 HourIncubation SIB-1 FDA - Presumptive Confirmed BAM Chi Relative MatrixStrain N^(a) Pos. Pos. Positive Square^(b) Sensitivity^(c) Plastic S.Newport 5 0 0 0 — — 20 11 11 13 0.406 84.6% Low level 20 20 20 20 0 100% High level Sealed S. Anatum 5 0 0 0 — — concrete 20 7 7 8 0.10487.5% Low level 20 20 20 20 0  100% High level Ceramic S. 5 0 0 0 — —tile Abaetetuba 20 10 10 7 0.898 142.9%  Low level 20 20 20 20 0  100%High level Stainless S. 5 0 0 0 — — steel^(d) Typhimurium: 20 0 0 2 2.050 10X C. Low freundii level 20 5 5 3 0.609 166.7%  High level ^(a)N =Number of test portions ^(b)Chi Square = Mantel-Haenszel: χ2 = (n −1)(ad − bc)²/[(a + b)(a + c)(b + d)(c + d)], where n = total number ofsamples tested by the two methods, a = number of samples positive by thetest method, b = number of samples negative by the test method, c =number of samples positive by the reference method and d = number ofsamples negative by the reference method ^(c)Relative sensitivity = a/c,where a = number of samples confirmed positive by the test method and c= number of samples positive by the reference method ^(d)Trial performedat the independent laboratory

Example 1.12.7: Ruggedness Studies

Ruggedness parameters studied were: incubation times (28 hours, 32 hoursand 46 and 50 hours) and incubation temperatures (34 and 40±1° C.). Twopositive controls (S. Abaetetuba and S. Anatum) and one negative control(E. coli) were tested in 5 replicates at the 3 log CFU/mL for Salmonellaserovars and at 7 log CFU/mL for E. coli. These tests were done ondifferent days as recommended. The summary of the results is given inTables 1-4 and 1-5.

TABLE 1-4 Temperature Variability 24 Hour 24 Hour 24 Hour 48 Hour 48Hour 48 Hour Strain 34° C. 37° C. 40° C. 34° C. 37° C. 40° C. S.Abaetetuba A + + + + + + B + + + + + + C + + + + + + D + + + + + +E + + + + + + S. Anatum A + + − + + + B + + − + + + C + + − + + + D + +− + + + E + + − + + + E. coli A − − − − − − B − − − − − − C − − − − − −D − − − − − − E − − − − − −

TABLE 1-5 Time Variability Strain 18 Hour 24 Hour 52 Hour S. AbaetetubaA + + + B + + + C + + + D + + + E + + + S. Anatum A + + + B + + +C + + + D + + + E + + + E. coli A − − − B − − − C − − − D − − − E − − −

Example 1.13: Lot-to-Lot Variability and Shelf Stability

Lot to lot variability studies were done in 5 replicates per level ofeach microorganism (S. Abaetetuba ATCC 35640, S. Anatum ATCC 9270 and E.coli ATCC 14948) for each of the three production lots tested (Table4a). One hundred microliters of culture dilutions were inoculated ontothe tip of a 3M™ ENVIROSWAB environmental sample collecting swab, whichwas then fully submerged into 15 mL of SIB-1 in its original sterilecontainer and incubated at 37° C. for 48 hours. The summary of theresults is given in Tables 1-6 and 1-7.

TABLE 1-6 Lot Variability Strain Lot 06007 Lot 06009 Lot 06011 S.Abaetetuba A + + + B + + + C + + + D + + + E + + + S. Anatum A + + +B + + + C + + + D + + + E + + + E. coli A − − − B − − − C − − − D − − −E − − −

TABLE 1-7 Shelf Stability Lot 06007 Lot 06007 Lot 06007 Strain 30 Days60 days 90 days S. Abaetetuba A + + + B + + + C + + + D + + + E + + + S.Anatum A + + + B + + + C + + + D + + + E + + + E. coli A − − − B − − − C− − − D − − − E − − −

Example 1.14: Analysis of Additional Real Environmental Samples

Environmental samples were obtained from food and non-food contactsurfaces in a coffee processing facility. Fifteen milliliters of SIB-1was added to the samples and incubated at 37° C. for 48 hours and theSIB reaction was recorded. All samples were streaked onto twodifferential selective agar media: (1) xylose lysine tergitol-4 (XLT)agar medium (see SS in Table 5 below); and (2) bismuth sulfide agarmedium (see BS in Table 5 below)). Salmonella-like colonies were pickedfor biochemical characterization using the MICROGEN™ GN-ID test(Microgen Bioproducts Ltd., Surrey, UK). All SIB-negative samples wereconfirmed to be negative on the differential selective agar media. Thepresumptive positive samples in the SIB yielded confirmed Salmonellaspecies and two false positive samples, Citrobacter freundii and E. coliinactive. The data are shown in Table 1-8:

TABLE 1-8 Growth and Fermentation of Environmental Samples in SIB-1Sample ID SIB Rxn SS Agar BS Agar GNID(Microgen) C1 + Lactose +C.freundii C2 − NG NG C3 − NG NG C4 − NG NG C5 + Lactose −, H2S +Salmonella spp. C6 + Lactose −, H2S + S.arizona C7 + Lactose −, H2S +S.arizona C8 − NG NG C9 − NG NG C10 + Lactose + + E. coli inactive C11 −NG NG C12 − NG NG C13 − NG NG C14 − NG NG C18 − NG NG C19 − NG NG C20 −NG NG C21 − NG NG C23 + Lactose w + C.freundii NG = No growth. Lactose −= Lactose non-fermenter. Lactose + = Lactose fermenter. Lactose w =Lactose weak fermenter. H₂S = Hydrogen sulfide producer.

Example 1.15: Discussion

SIB-1 is an easy to use and interpret screening test for Salmonellaspecies in environmental samples. Inclusivity and exclusivity studiesrevealed that SIB-1 is very comprehensive for the detection ofSalmonella species at very low levels (10-100 CFU/sample). Two strains,S. Cubana and S. Gallinarum, were originally negative in the inclusivitystudy. An additional isolate of S. Cubana was obtained from a differentsource, the University of Pennsylvania Salmonella Reference Center. Thisparticular isolate was positive in fermenting the indicator compound inSIB-1 in contrast to the isolate obtained from the University ofMinnesota's culture collection. These data suggest that the Minnesotaisolate was likely defective in a metabolic pathway for fermentation ofthe indicator compound. In regard to specificity, high levels of someCitrobacter species remain to be a possible source of false positiveresults. False positive results, although not desired by the typical enduser, still tells a great deal about the overall microbial cleanlinessof the areas sampled. Learning about the presence of Citrobacter speciesis important for another aspect, since many Citrobacter species occupysimilar niches to Salmonella species and arise as contamination sourcesfrom the GI tracts of warm blooded animals. This information ispotentially useful when monitoring food processing surfaces intended tobe free of microflora after sanitation operations. These resultsdemonstrate a cross reaction in two of three Citrobacter species testedunderscoring the need to confirm all SIB-1-positive results by thetraditional biochemical or genetic methods.

SIB-1 was found to be at least as sensitive as the reference method inall the surfaces studied. In fact SIB-1 was slightly more sensitive thanthe reference method in one of the method comparison studies: fivepositives for SIB-1 versus three positives for the FDA-BAM method in thestainless steel study.

Regarding the ruggedness studies, recommended parameters have beenstudied for SIB-1. Results of the ruggedness studies suggested that forthe most part, selected deviations from test parameters did notinterfere with the true detection of microorganisms selected with theexception of Salmonella Anatum, which was not detected at 40° C. at 24hours incubation.

Lot-to-lot variability studies showed that there was no differencebetween the production lots. Shelf life studies documented in Table 4brevealed that SIB-1 is shelf-stable at 3 months of refrigerated storage.

In conclusion, SIB-1 is a unique, easy to perform rapid detection testfor Salmonella species in environmental samples. The test has beendemonstrated to be substantially equivalent to the FDA-BAM method forthe four selected surfaces as well as both sufficientlySalmonella-inclusive and exclusive with the notable exception of someCitrobacter species. These studies detail the utility of SIB-1 asdiagnostic screening test. Since SIB-1 is a self-contained test, itminimizes cross contamination. Furthermore, this study shows that SIB-1is compatible with the FDA-BAM confirmation methods. This compatibilityprovides a more economical solution to Salmonella screening without anycompromise in sensitivity. It is expected that SIB-1 will enable moreon-site Salmonella monitoring in the environmental samples and therebycontribute to the overall goal of raising food safety standards in theprocessing environment.

Example 2. SIB-1 Supplemented with Doxycycline

To increase the specificity of SIB-1, SIB-1 was supplemented with 0.005g/L doxycycline. Sponges were inoculated at 10-100 CFU/Sample. Theculture response of twenty Salmonella enterica serovars was recordedafter 48 hours incubation at 37° C. The results are shown in Table 2-1.

TABLE 2-1 Inclusivity of SIB-1 Supplemented with Doxycycline SalmonellaIndicator Broth Salmonella enterica serovar Reaction S. Aberdeen + S.Aequatoria + S. Alabama + S. Altendorf + S. Austin + S. Ball + S.Berkeley + S. Brookfield + S. California + S. Canastel + S. Carmel + S.Carrau + S. Champaign + S. Chicago + S. Colombo + S. Ealing + S.Dahlem + S. Gallinarum + S. Houten + S. Kottbus +These results show that adding doxycycline to SIB-1 does not inhibitSalmonella spp. growth.

Example 3: SIB-1 Supplemented with Rifampicin

To increase the specificity of SIB-1, SIB-1 was supplemented with 0.002g/L rifampicin. Sponges were inoculated at 10-100 CFU/Sample. Theculture response of twenty Salmonella enterica serovars was recordedafter 48 hours incubation at 37° C. The results are shown in Table 3-1:

TABLE 3-1 Inclusivity of SIB-1 Supplemented with Rifampicin SalmonellaIndicator Broth Salmonella enterica serovar Reaction S. Illinois + S.Lexington + S. Manchester + S. Minnesota + S. Mississippi + S. Napoli +S. Pensacola + S. Pretoria + S. Shanghai + S. Sunsvall + S. Carmel + S.Carrau + S. Champaign + S. Chicago + S. Colombo + S. Ealing + S.Dahlem + S. Gallinarum − S. Houten + S. Kottbus + S. Johannesberg + S.Oranienberg + S. Thomasville + S. Brandenburg + S. Manhattan + S.Urbana + S. Stanley + S. Panama + S. Cerro + S. Hadar +These results show that supplementing SIB-1 with rifampicin does notinhibit Salmonella spp. growth.

Example 4. Antibiotic-Supplemented Basal SIB with and without NMP

To demonstrate the increased specificity of EPI-supplemented medium,four non-Salmonella species giving positive reactions in basal SIB orSIB-1 were tested in doxycycline-supplemented (0.005 g/L doxycycline),rifampicin-supplemented (0.002 g/L rifampicin), orlinezolid-supplemented (0.005 g/L linezolid) basal SIB with and without0.2 mM (0.044 g/L) 1-(1-naphthylmethyl)piperazine (NMP). The cultureswere grown to stationary phase in BHI medium overnight at 37° C. andserially diluted out 3-log orders to obtain suspensions withapproximately 10⁵-10⁶ CFU/mL. One tenth milliliter was pipetted onto asterile collection sponge (3M™ ENVIROSWAB) and 15 mL of the basal SIBsupplemented with either antibiotic alone or antibiotic with NMP wasadded. The samples were incubated for 48 hours at 37° C. and the colorreactions were recorded. The results are shown in Tables 4-1 through4-3:

TABLE 4-1 Exclusivity of Doxycyline-Supplemented Basal SIB with andwithout NMP Salmonella Indicator Salmonella Indicator Broth ReactionBroth Reaction Basal Media + Basal Media + Species DoxycyclineDoxycycline/NMP Enterobacter + − aerogenes Enterobacter cloacae + −Citrobacter freundii + + Citrobacter koseri + +

TABLE 4-2 Exclusivity of Rifampicin-Supplemented Basal SIB with andwithout NMP Salmonella Indicator Salmonella Indicator Broth ReactionBroth Reaction Basal Media + Basal Media + Species RifampicinRifampicin/NMP Enterobacter + − aerogenes Enterobacter cloacae + −Citrobacter freundii + − Citrobacter koseri + −

TABLE 4-3 Exclusivity of Linezolid-Supplemented Basal SIB with andwithout NMP Salmonella Indicator Salmonella Indicator Broth ReactionBroth Reaction Basal Media + Basal Media + Species LinezolidLinezolid/NMP Enterobacter + − aerogenes Enterobacter cloacae + −Citrobacter freundii + − Citrobacter koseri + −These results show that the effectiveness of the EPI in enhancing theselectivity of Salmonella spp. selective agents.

Example 5: Demonstration of Salmonella-Selective EPI Activity of4-Chloroquinoline

To increase the specificity of basal SIB, basal SIB was supplementedwith 4-chloroquinoline (4CQ) at a final concentration of 0.2 mM (0.023g/L) and 0.002 g/L rifampicin. Sponges were inoculated at 10-100CFU/Sample. The culture response of twenty Salmonella enterica serovarswas recorded after 48 hours incubation at 37° C. The data are shown inTable 5-1:

TABLE 5-1 Inclusivity of Basal SIB Supplemented with Rifampicin and 4CQSalmonella Indicator Broth Salmonella enterica serovar Reaction S.Aberdeen + S. Aequatoria + S. Alabama + S. Altendorf + S. Austin + S.Ball + S. Berkeley + S. Brookfield + S. California + S. Canastel + S.Carmel + S. Carrau + S. Champaign + S. Chicago + S. Colombo + S.Ealing + S. Dahlem + S. Gallinarum + S. Houten + S. Kottbus +These results show that supplementing basal SIB with rifampicin and4-chloroquinoline does not inhibit Salmonella spp. growth.

Example 6: Antibiotic-Supplemented Basal SIB with and without 4CQ

To further demonstrate the increased Salmonella-selective specificity ofthe EPI- and antibiotic-supplemented SIB, four non-Salmonella speciesgiving positive reactions in basal SIB or SIB-1 were tested in therifampicin-supplemented media (0.002 g/L rifampicin) with and without4-chloroquinoline (4CQ). The cultures were grown to stationary phase inBHI medium overnight at 37° C. and serially diluted out 3-log orders toobtain suspensions with approximately 10⁵-10⁶ CFU/mL. One tenthmilliliter was pipetted onto a sterile collection sponge (3M™ENVIROSWAB) and 15 mL of the basal SIB supplemented with eitherantibiotic alone or antibiotic with 4CQ was added. The samples wereincubated for 48 hours at 37° C. and the color reactions were recorded.The data are shown in Table 6-1:

TABLE 6-1 Exclusivity of Rifampicin-Supplemented Basal SIB with andwithout 4CQ Salmonella Indicator Salmonella Indicator Broth ReactionBroth Reaction Basal Media + Basal Media + Species RifampicinRifampicin/4CQ Enterobacter + − aerogenes Enterobacter cloacae + −Citrobacter freundii + − Citrobacter koseri + −These results show that the effectiveness of 4CQ in enhancing theselectivity of Salmonella spp. selective agents.

Example 7: Discussion

Several media formulations were tested for permitting growth ofSalmonella while inhibiting growth of non-Salmonella species. Initialmedia formulations lacking myricetin, antibiotics, and EPIs did noteffectively inhibit the growth of some cross-reacting species, notably,K. ozaenae, E. aerogenes, C. freundii, C. koseri and E. coli inactive.To minimize the potential of such environmental microflora to contributeto false positive reactions in selective indicator media, flavonoidcompounds were investigated. Myricetin was tested as a potentialsuitable inhibitor for at least some of these gram negative species.Myricetin was observed to be effective against K. oxytoca and E.aerogenes but was of little use against the Citrobacter and E. coliinactive isolates. Media supplemented with the halo-quinoline4-chloroquinoline (4CQ) or the naphthylmethyl piperazine1-(1-naphthylmethyl)piperazine (NMP) in combination with certainantibiotics were substantially effective in preventing outgrowth of allthe E. coli inactive isolates and some of the Citrobacters, particularlyC. freundii.

Particularly effective media formulations included the componentscontained in the basal selective indicator broth discussed below(including myricetin), an EPI (i.e., either halo-quinolines such as4-chloroquinoline or naphthylmethyl piperazines such as1-(1-naphthylmethyl)piperazine), and tetracycline, doxycycline,rifampicin, or linezolid. Such media were capable of controlling most ofthe gram negative competitors found in the environment, excluding someC. koseri strains. Compositions including rifampicin in place oftetracycline or doxycycline were highly inhibitory to all theCitrobacter species in the culture collection.

The data collectively demonstrate the enhancement of antimicrobialactivity of selective agents in the presence of the EPIs. As shownherein, these agents effectively inhibit the growth of gram-negativecompetitors of Salmonella while not inhibiting the growth and metabolismof Salmonella.

Example 8: Selection Shiga Toxin-Producing E. coli from Commensal E.coli Example 8.1: Background

The examples above show selective enrichment media for isolation anddetection of Salmonella species. The examples that follow show use ofsimilar compositions for the selection of Shiga toxin-producing E. coli(STEC). Since the late 1980's, commercial products have been developedfor the isolation and detection of E. coli O157:H7 due the widespreadfood contamination events with this pathogen. Proliferation of otherpathogenic E. coli serotypes has prompted regulators to requirescreening for a wider array of STEC species. Dubbed the “big Six”serotypes, E. coli strains O26, O111, O45, O145, O121, O103 in additionto O157 must now be screened for in meat processing within the UnitedStates. There remains no effective selective enrichment mediumsufficiently restrictive to most other gram negative bacteria butpermissive to growth of these E. coli serotypes.

Example 8.2: SIB-2

A variant of SIB-1, referred to as SIB-2, was generated. The compositionof SIB-2 is shown in Table 8-1.

TABLE 8-1 SIB-2 Composition Component Concentration Peptone 10 g/L MgCl₂hexahydrate 13 g/L Bromocresol purple 0.02 g/L Sulfanilamide 1 g/LSulfathiazole 0.1 g/L NIAPROOF ® 4 2 mL/L (2.12 g/L) 2-Deoxy-D-ribose 5g/L Cycloheximide 0.05 g/L Ascorbic acid 1 g/L p-Bromobenzoic acid 0.04g/L Novobiocin 0.02 g/L Brilliant green 0.001 g/L Myricetin 0.03 g/LNaphthylmethyl piperazine (NMP) 0.044 g/L Doxycycline 0.005 g/L

Example 8.3: Inclusivity and Exclusivity Studies

Shiga toxin-producing E. coli (STEC) cultures were obtained from thePenn State University E. coli Reference Center. Determinations for thepresence of the Shiga toxin genes (stx1 and stx2) were conducted at thePenn State University E. coli Reference Center. Serial dilutions fromovernight grown pure cultures of STEC strains were made into sterile0.1% peptone water. One hundred microliters from log −8 dilution(˜1.0E+3 CFU/mL) were plated onto brain heart infusion (BHI) plates forestimating the number of cells tested. For inclusivity studies, 0.1 mL(estimated cell amount ranging from 10 to 100 CFU of STEC) of a dilutionaliquot was aseptically transferred onto the top of the sampling sponge.For exclusivity studies, 0.2 mL (estimated cell amount >1,000,000 CFU ofnon-STEC) of a dilution aliquot was aseptically transferred onto the topof the sampling sponge. The sampling sponge was then placed back intothe sterile tube and submerged in 15 mL of SIB-2 media. The growth ofthe STEC and non-STEC strains is shown in Table 8-2.

TABLE 8-2 E. coli Strain Growth in SIB-2 Media Cultural response2-DeoxyD- E. coli Strain Shiga Toxin in SIB Ribose O104:H4 ND(presumptive +) G − O26:H1 + G − O103:H2 + G − O91:H21 ND (presumptive+) G − O157:H7 + G − O145:H28 + G − O111:H8 + G + ATCC 13706 ND(presumptive −) NG ATCC 14948 ND (presumptive −) NG NCIMB 11943 ND(presumptive −) NG NCTC 10538 ND (presumptive −) NGTable 8-2 documents the ability of seven different STEC serovars to growin the presence of the selective agents present in SIB-2 media. Bycontrast none of the commensal E. coli strains inoculated atsubstantially higher inocula levels were capable of growth in SIB media.Most of the active STEC strains were incapable of fermenting theincorporated 2-deoxy D-ribose in the media. A more suitable fermentablesubstrate would likely be lactose because most E. coli strains areactive lactose fermenters while most Salmonella species are negative forlactose fermentation.

Example 8.4: Selection of STEC from Bovine Manure in Feed Lots

To further expand the data set on the relative selectivity of the SIB-2media for Shiga toxin-producing E. coli strains versus commensal E.coli, a series of E. coli isolates originally obtained from bovinemanure in feed lots were examined. The isolates were obtained from Dr.Francisco Diez-Gonzalez, Department of Food Science and Nutrition,University of Minnesota. Table 8-3 provides a summary of the strainisolate biotypes.

TABLE 8-3 E. coli Provenance E. coli Strain Shiga Toxin Origin O157:H7 +Penn State E. coli Ref. Center O145:H28 + Penn State E. coli Ref. CenterO104:H4 ND (presumptive +) Penn State E. coli Ref. Center O26:H1 + PennState E. coli Ref. Center O111:H8 + Penn State E. coli Ref. CenterO103:H2 + Penn State E. coli Ref. Center O91:H21 ND (presumptive +) PennState E. coli Ref. Center 261 − University of MN K12-W3110 − Universityof MN A11-3 ND (presumptive −) University of MN DH5-a − University of MN3TF1 − University of MN 3BF2 ND (presumptive −) University of MN CR63 −University of MN

Serial dilutions from overnight-grown pure cultures of the E. colistrains were made into sterile 0.1% peptone water. One hundredmicroliters from log −8 dilution (˜1.0E+3 CFU/mL) were plated onto BHIplates for estimating the number of cells tested. Stock BHI cultureswere diluted serially into 0.2 mL of SIB-2 media to obtain 8 logdilutions yielding an initial inoculation concentration of between 100to 1000 CFU/mL. Cultures were grown at 37° C. for 18 hours. Highestdilutions were streaked onto either BHI or selective differential agarsfor cell growth estimates.

Table 8-4 summarizes the cultural response of E. coli strains enrichedin SIB-2. SIB-2 cultures were streaked onto BHI for growth estimates.From the data in Table 8-4 the SIB-2 medium permits growth of all theSTEC isolates at low initial inoculation (<10³ CFU/mL). By contrast themedium permits only three of the seven commensal E. coli strains fromgrowing from low initial inoculations.

TABLE 8-4 Growth of E. coli Strains in SIB-2 Broth E. coli Strain SIBGrowth O157:H7 + O145:H28 + O104:H4 + O26:H1 + O111:H8 + O103:H2 +O91:H21 + 261 − K12-W3110 − A11-3 + DH5-a + 3TF1 + 3BF2 − CR63 +

Example 8.5: Selection of STEC with SIB-2 Supplemented withNitrofurantoin

To examine for the potential development of a more STEC-selective mediumthan SIB-2, we supplemented SIB-2 with 0.005 mg/L of nitrofurantoin, abroad spectrum antibiotic frequently used to treat E. coli urinary tractinfections.

Table 8-5 summarizes the growth response of E. coli strains in SIB-2supplemented with 0.005 g/L nitrofurantoin. As can be seen from the datain Table 8-5, supplementation of SIB-2 with nitrofurantoin renders themedia more selective with respective to STEC strains. Whereas SIB-2alone permitted growth of four of seven commensal E. coli strains, SIB-2supplemented with nitrofurantoin excluded all but one of the commensalE. coli strains.

TABLE 8-5 Growth of E. coli Strains in SIB-2 Broth Supplemented withNitrofurantoin E. coli Strain SIB-2 Growth O157:H7 + O145:H28 +O104:H4 + O26:H1 + O111:H8 + O103:H2 + O91:H21 + 261 − K12-W3110 − A11-3− DH5-a − 3TF1 + 3BF2 − CR63 −

Example 9: Co-Selection of Salmonella and Shiga Toxin-Producing E. coli

An important trend in the meat industry in the United States is theability to cultivate E. coli and Salmonella in the same selectiveenrichment media. Laboratories would like to streamline their processesby reducing the steps to regulatory compliance to one operation forscreening for both E. coli and Salmonella. The examples above show theability of the media of the invention to enrich for Shigatoxin-producing E. coli and Salmonella independently. The presentexample shows the ability of the media to support the growth of bothpathogens through co-cultivation.

S. abetetuba obtained from the University of Pennsylvania SalmonellaReference Center was cultured in BHI overnight to obtain ˜10¹⁰ CFU/mLstock cultures. A comparable culture from each of the seven STEC strainsshown in Tables 8-2 and 8-3 were also prepared and mixed with an equalvolume (0.25 mL) of the Salmonella culture. An aliquot (0.02 mL) of eachmixed culture was serially diluted out in SIB-2 eight log orders toobtain initial populations of 10² to 10³ CFU/mL of each pathogen. TheSIB-2 cultures were incubated for eighteen hours at 37° C. and streakedon an agar medium based on a variant of SIB-2 having the compositiondepicted in Table 9-1. The agar medium was supplemented with5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) and isopropylβ-D-1-thiogalactopyranoside (IPTG) to permit identification of the E.coli colonies (blue) from the Salmonella colonies (white). Growthresults are summarized in Table 9-2.

TABLE 9-1 Agar Medium Composition Component Concentration Peptone 10 g/LMgCl₂ hexahydrate 13 g/L Sulfanilamide 1 g/L NIAPROOF ® 4 2 mL/L (2.12g/L) Cycloheximide 0.05 g/L Novobiocin 0.02 g/L Brilliant green 0.001g/L Myricetin 0.005 g/L Naphthylmethyl piperazine 0.044 g/L Doxycycline0.005 g/L Nitrofurantoin 0.005 g/L X-gal 0.05 g/L IPTG 0.02 g/L Agar 15g/L

TABLE 9-2 Growth Results of Co-Cultivation E. coli Strain GrowthO157:H7 + O145:H28 − O104:H4 + O26:H1 + O111:H8 + O103:H2 + O91:H21 −The data show that after 24 hours of cultivation on the agar medium,five of seven STEC strains grew among a predominant population of S.abetetuba. After a further 24 hours, detectable blue colonies could beseen on both the O145 and O91 co-cultures in the presence of Salmonellabackground.

What is claimed is:
 1. A selective enrichment medium comprising: aselective agent comprising one or more of a sulfa drug, a surfactant, anaminocoumarin, cycloheximide, ascorbic acid, bromobenzoic acid,myricetin, and 2-deoxy-D-ribose and one or more of a rifamycin, apolyketide, and an oxazolidinone; and an efflux pump inhibitorcomprising one or more of 1-(1-naphthylmethyl)piperazine and4-chloroquinoline, wherein the selective agent and the efflux pumpinhibitor are present in amounts effective to enrich for a microorganismselected from the group consisting of Salmonella and Shigatoxin-producing E. coli.
 2. The selective enrichment medium of claim 1wherein the efflux pump inhibitor comprises1-(1-naphthylmethyl)piperazine.
 3. The selective enrichment medium ofclaim 1 wherein the efflux pump inhibitor comprises 4-chloroquinoline.4. The selective enrichment medium of claim 1 wherein the selectiveagent comprises a sulfa drug, wherein the sulfa drug comprises one ormore of sulfanilamide and sulfathiazole.
 5. The selective enrichmentmedium of claim 1 wherein the selective agent comprises a surfactant,wherein the surfactant comprises 7-ethyl-2-methyl-4-undecyl sulfate or asalt thereof.
 6. The selective enrichment medium of claim 1 wherein theselective agent comprises an aminocoumarin, wherein the aminocoumarincomprises novobiocin.
 7. The selective enrichment medium of claim 1wherein the selective agent further comprises a supravital stain,wherein the supravital stain comprises brilliant green.
 8. The selectiveenrichment medium of claim 1 wherein the selective agent comprises arifamycin, wherein the rifamycin comprises rifampicin.
 9. The selectiveenrichment medium of claim 1 wherein the selective agent comprises apolyketide, wherein the polyketide comprises doxycycline.
 10. Theselective enrichment medium of claim 1 wherein the selective agentcomprises an oxazolidinone, wherein the oxazolidinone compriseslinezolid.
 11. The selective enrichment medium of claim 1 wherein theselective agent comprises a sulfa drug, a surfactant, an aminocoumarin,cycloheximide, and myricetin, and one or more of a rifamycin, apolyketide, and an oxazolidinone.
 12. The selective enrichment medium ofclaim 1 further comprising a visual indicator that indicates thepresence of a microorganism selected from the group consisting ofSalmonella and E. coli.
 13. The selective enrichment medium of claim 1wherein the selective agent and the efflux pump inhibitor are present inamounts effective to inhibit growth of at least one non-Salmonellaspecies to a greater extent than one or more Salmonella species.
 14. Theselective enrichment medium of claim 1 wherein the selective agent andthe efflux pump inhibitor are present in amounts effective to inhibitgrowth of at least one non-Shiga toxin-producing E. coli strain to agreater extent than one or more Shiga toxin-producing E. coli strains.15. The selective enrichment medium of claim 1 further comprising acarbon and nitrogen source and an inorganic salt.
 16. The selectiveenrichment medium of claim 13 wherein the selective agent comprises asulfa drug, a surfactant, an aminocoumarin, cycloheximide, andmyricetin, and one or more of a rifamycin, a polyketide, and anoxazolidinone.
 17. The selective enrichment medium of claim 14 whereinthe selective agent comprises a sulfa drug, a surfactant, anaminocoumarin, cycloheximide, and myricetin, and one or more of arifamycin, a polyketide, and an oxazolidinone.
 18. The selectiveenrichment medium of claim 1, wherein: the selective agent comprises: asulfa drug in an amount from about 0.1 g/L to about 10 g/L; a surfactantin an amount from about 0.2 g/L to about 22.5 g/L; an aminocoumarin inan amount from about 0.004 g/L to about 0.5 g/L; cycloheximide in anamount from about 0.002 g/L to about 0.5 g/L; ascorbic acid in an amountfrom about 0.1 g/L to about 10 g/L; bromobenzoic acid in an amount fromabout 0.002 g/L to about 0.5 g/L; myricetin in an amount from about0.002 g/L to about 0.5 g/L; and a polyketide in an amount from about0.0002 g/L to about 0.1 g/L; and the efflux pump inhibitor comprises1-(1-naphthylmethyl)piperazine.
 19. The selective enrichment medium ofclaim 18, wherein: the sulfa drug comprises one or more of sulfanilamideand sulfathiazole; the surfactant comprises 7-ethyl-2-methyl-4-undecylsulfate or a salt thereof; the aminocoumarin comprises novobiocin; andthe polyketide comprises one or more of tetracycline and doxycycline.20. A method of selectively enriching for a microorganism selected fromthe group consisting of a Salmonella species and a Shiga toxin-producingE. coli strain, comprising culturing a sample suspected of containingthe microorganism in the selective enrichment medium of claim
 1. 21. Themethod of claim 20 wherein the culturing grows a Salmonella species, aShiga toxin-producing E. coli strain, or both a Salmonella species and aShiga toxin-producing E. coli strain.
 22. The method of claim 20 furthercomprising detecting presence of the microorganism.